Fc-free antibodies comprising two fab-fragments and methods of use

ABSTRACT

The present invention relates to bispecific antibodies comprising at least two Fab fragments, wherein the first Fab fragment comprises at least one antigen binding site specific for a first antigen; and the second Fab fragment comprises at least one antigen binding site specific for a second antigen, wherein either the variable regions or the constant regions of the second Fab heavy and light chain are exchanged and the two Fab fragments are connected to each other by two peptide linkers; and wherein the bispecific antibody is devoid of a Fc domain; methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 9, 2015, is named P31270-US_SL.txt and is 205,458 bytes in size.

FIELD OF THE INVENTION

The present invention relates to bispecific antibodies comprising at least two Fab fragments, wherein the first Fab fragment comprises at least one antigen binding site specific for a first antigen; and the second Fab fragment comprises at least one antigen binding site specific for a second antigen, wherein either the variable regions or the constant regions of the second Fab heavy and light chain are exchanged and the two Fab fragments are connected to each other by two peptide linkers; and wherein the bispecific antibody is devoid of a Fc domain; methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

The present invention further relates to bispecific antibodies that specifically bind a T-cell activating antigen and a Tumor Antigen (TA), comprising a first Fab fragment and a second Fab fragment, wherein either the variable regions or the constant regions of the second Fab heavy and light chain are exchanged and the two Fab fragments are connected to each other by two peptide linkers; and wherein the bispecific antibody does not comprise a Fc domain; methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

BACKGROUND

Monoclonal antibodies (mAbs) are an increasingly important class of therapeutic agents. Apart from mAb products composed of the full-size form of IgG, a wide variety of multispecific recombinant antibody formats have been developed, e.g. tetravalent bispecific antibodies by fusion of, e.g., an IgG antibody format and single chain domains (see e.g. Coloma, M. J., et al., Nature Biotech 15 (1997) 159-163; WO 2001/077342; and Morrison, S. L., Nature Biotech 25 (2007) 1233-1234).

Several other new formats wherein the antibody core structure (IgA, IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- or tetrabodies, minibodies, several single chain formats (scFv, Bis-scFv), which are capable of binding two or more antigens, have been developed (Holliger, P., et al., Nature Biotech 23 (2005) 1126-1136; Fischer, N., Léger, O., Pathobiology 74 (2007) 3-14; Shen, J., et al., Journal of Immunological Methods 318 (2007) 65-74; Wu, C., et al., Nature Biotech. 25 (2007) 1290-1297).

All such formats use linkers either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fuse e.g. two Fab fragments or scFvs (Fischer, N., Léger, O., Pathobiology 74 (2007) 3-14). Tandem scFVs are two scFv fragments linked by an extra peptide linker and are also referred to as (scFv)2. By reducing the length of the peptide linker between the variable domains, Diabodies are generated. Adding an extra peptide linker between the two polypeptides yields so called single chain diabodies. US 2007/0274985 relates to antibodies comprising single chain Fab (scFab) fragments. Antibody fragments have both pros and cons as therapeutics compared with full-size monoclonal antibodies: One advantage is that they are smaller and penetrate tissues and tumors more rapidly. In addition, the small size of fragments has been suggested to permit binding to epitopes not accessible to full-sized monoclonal antibodies. On the downside, fragments demonstrate short circulating half-lives in humans, likely due to kidney clearance. The shorter half-life may prevent sufficient accumulation of therapy at the targeted site. Production of antibody fragments is not trivial, as fragments are likely to form aggregates and can be less stable than full-size monoclonal antibodies. In addition, unwanted pairing of noncongnate heavy and light chains results in formation of inactive antigen-binding sites and/or other non-functional undesired side-products, which is a major problem in clinical-scale production and therapeutic application of antibody fragments. In particular Tandem-Fab constructs where two or more Fabs are fused with each other via one connector are not feasible due to the random association of the two light chains resulting in inactive, undesired side products. These drawbacks have now been overcome with the new antibody format of the invention. Provided therein is a new bispecific antibody format that can be easily produced with an increased yield due to a decreased amount of mispaired side-products, which show less aggregation than bispecific antibody fragments known in the art. Using the crossover approach correct LC association can be enforced without the need for the generation of a common light chain. Common light chan approach is not possible for existing antibodies. Further, the new bispecific antibody format is stable and does not aggregate at higher temperatures than existing bispecific formats, which allows better producability and developability. In addition, the new bispecific antibody format has a higher molecular weight compared to many conventional bispecific antibody fragments, thus preventing excessive kidney clearance and leading to an improved half-life in vivo. The new bispecific antibody format is fully functional and has comparable or improved binding and activity as corresponding conventional bispecific antibodies.

The selective destruction of an individual cell or a specific cell type is often desirable in a variety of clinical settings. For example, it is a primary goal of cancer therapy to specifically destroy tumor cells, while leaving healthy cells and tissues undamaged. One approach is to selectively induce an immune response against the tumor, which triggers the attack and subsequent destruction of tumor cells by immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs). CTLs constitute the most potent effector cells of the immune system, however they cannot be activated by the effector mechanism mediated by the Fc domain of conventional therapeutic antibodies. In this regard, bispecific antibodies which are able to bind to a surface antigen on cancer cells and to an activating invariant component of the cell receptor (TCR) complex have become of interest in recent years. The simultaneous binding of the bispecific antibody to both of its targets forces a temporary interaction between cancer cell and T cell, causing activation of cytotoxic T cells and subsequent lysis of the tumor cell.

Several bispecific antibody formats have been developed and their suitability for T cell mediated cancer immunotherapy investigated. Out of these, the so-called BiTE (bispecific T cell engager) molecules have been very well characterized and already shown some promising results in the clinic (reviewed in Nagorsen and Baiuerle, Exp Cell Res 317, 1255-1260 (2011)). BiTEs are tandem scFv molecules wherein two scFv molecules are fused by a flexible linker. Further bispecific formats being evaluated for T cell engagement include diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies (Kipriyanov et al., J Mol Biol 293, 41-66 (1999)). A more recent development are the so-called DART (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117, 4542-51 (2011)). The so-called triomabs, which are whole hybrid mouse/rat IgG molecules and also currently being evaluated in clinical trials, represent a larger sized format (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)).

However, the bispecific antibodies developed for T cell mediated cancer immunotherapy known so far have major drawbacks relating to their efficacy, toxicity and applicability. Small constructs such as, for example, BiTE molecules—while being able to efficiently crosslink effector and target cells—have a very short serum half life requiring them to be administered to patients by continuous infusion. IgG-like formats on the other hand—while having the great benefit of a long half life—suffer from toxicity associated with the native effector functions inherent to IgG molecules. This immunogenic potential constitutes another unfavorable feature of IgG-like bispecific antibodies, for successful therapeutic development. Finally, a major challenge in the general development of bispecific antibodies remains the production of bispecific antibody constructs at a clinically sufficient quantity and purity. The mispairing of antibody heavy and light chains of different specificities upon co-expression, decreases the yield of the correctly assembled construct and results in a number of non-functional side products.

Given the difficulties and disadvantages associated with currently available bispecific antibodies for T cell mediated cancer immunotherapy, there remains a need for novel, improved formats of such molecules. These drawbacks have now been overcome with the new bispecific antibodies of the invention. The new bispecific antibodies can be easily produced, with an increased yield due to a decreased amount of mispaired side-products, which show less aggregation than bispecific antibody fragments known in the art. In addition, the new bispecific antibodies are stable at increased temperatures compared to bispecific antibody formats known in the art. Using the crossover approach correct chain association can be enforced without the need for the generation of a common light chain. In addition, the new the new bispecific antibodies has a higher molecular weight compared to many conventional bispecific antibody fragments, thus preventing excessive kidney clearance and leading to an improved half-life in vivo. Further, only two plasmids are needed to produce the new bispecific antibodies. The new bispecific antibodies are fully functional and have comparable or improved binding and activity as corresponding conventional bispecific antibodies.

The present invention provides bispecific antigen binding molecules designed for T cell activation and re-direction that combine good efficacy and produceability with low toxicity and favorable pharmacokinetic properties.

SUMMARY

The present invention relates to bispecific antibodies comprising at least two Fab fragments, wherein the first Fab fragment comprises at least one antigen binding site specific for a first antigen; and the second Fab fragment comprises at least one antigen binding site specific for a second antigen wherein either the variable regions or the constant regions of the second Fab heavy and light chain are exchanged and the two Fab fragments are connected to each other by two peptide linkers; and wherein the bispecific antibody is devoid of a Fc domain. Preferably said peptide linker is a (G4S)2 linker (SEQ ID NO: 28).

In one embodiment said antibody additionally comprises a third Fab fragment. In another embodiment said third Fab fragment comprises at least one antigen binding site specific for the first or second antigen, preferably for the first antigen.

In one embodiment the third Fab fragment is connected to the light chain or the heavy chain of the first Fab fragment. In another embodiment the third Fab fragment is connected to N or C-terminus of the light chain or the heavy chain of the second Fab fragment. In one embodiment the third Fab fragment is connected to the first or second Fab fragment via one or more peptide linker(s). Preferably said peptide linker is a (G4S)2 linker (SEQ ID NO: 28).

The bispecific antibodies according to the invention are at least bivalent and can be trivalent or multivalent e.g. tetravalent. In one embodiment said bispecific antibodies are bivalent (1+1 format) with one binding site each targeting a first antigen and a second antigen, respectively. In another embodiment said bispecific antibodies are trivalent (2+1 format) with two binding sites each targeting a first antigen and one binding site targeting a second antigen, as detailed in the following section.

The present invention relates to bispecific antibodies that specifically bind a T-cell activating antigen and a Tumor Antigen (TA), comprising a first Fab fragment and a second Fab fragment, wherein either the variable regions or the constant regions of the second Fab heavy and light chain are exchanged and the two Fab fragments are connected to each other by two peptide linkers and wherein the bispecific antibody does not comprise a Fc domain.

The antibodies of the invention specifically bind to a Tumor Antigen on the surface of a tumor cell and at the same time bind to T-cell activating antigen. By that the bispecific antibody is capable to elecit an immune response specifically at the site of the tumor, subsequently resulting in apoptosis of the target cell.

In one aspect, a bispecific antibody that specifically binds a T-cell activating antigen and a Tumor Antigen (TA) is provided, comprising at least twoFab fragments which are connected to each other by two peptide linkers, wherein the first Fab fragment comprises at least one antigen binding site specific for a Tumor Antigen (TA); and the second Fab fragment comprises at least one antigen binding site specific for a T-cell activating antigen, wherein either the variable regions or the constant regions of the second Fab heavy and light chain are exchanged; and wherein the bispecific antibody is devoid of a Fc domain.

In particular, the present invention relates to bispecific antibodies wherein the T-cell activating antigen is a CD3 T-Cell Co-Receptor (CD3) targeting antigen. In one embodiment the Tumor Antigen is selected from the group of Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), Carcinoembryonic Antigen (CEA), Fibroblast Activation Protein (FAP) and CD33. In one preferred embodiment the Tumor Antigen is MCSP.

In one aspect, a bispecific antibody that specifically binds CD3 T-Cell Co-Receptor (CD3) antigen and a Tumor Antigen (TA) is provided, comprising at least two Fab fragments which are connected to each other by two peptide linkers, wherein the first Fab fragment comprises at least one antigen binding site specific for a Tumor Antigen (TA); and the second Fab fragment comprises at least one antigen binding site specific for a CD3 T-Cell Co-Receptor (CD3) wherein either the variable regions or the constant regions of the second Fab heavy and light chain are exchanged; and wherein the bispecific antibody is devoid of a Fc domainPreferably said peptide linker is a (G4S)2 linker (SEQ ID NO: 28).

In one embodiment said antibody additionally comprises a third Fab fragment, which is connected to the first or second fragment by one or two peptide linkers. In another embodiment said third Fab fragment comprises at least one antigen binding site specific for a Tumor Antigen. In one embodiment the third Fab fragment is connected to the N or C-terminus of the light chain or the heavy chain of the first Fab fragment. In another embodiment the third Fab fragment is connected to the N or C-terminus of the light chain or the heavy chain of the second Fab fragment. In one embodiment the third Fab fragment is connected to the first or second Fab fragment by one or two peptide linkers. Preferably said peptide linker is a (G4S)2 linker (SEQ ID NO: 28)

The bispecific antibodies according to the invention are at least bivalent and can be trivalent or multivalent e.g. tetravalent or hexavalent. In one embodiment said bispecific antibodies are bivalent (1+1 format) with one binding site each targeting a Tumor Antigen (TA) and a T-cell activating antigen, respectively. In another embodiment said bispecific antibodies are trivalent (2+1 format) with two binding sites each targeting a Tumor Antigen (TA) and one binding site targeting a T-cell activating antigen, as detailed in the following section. In a preferred embodiment said a T-cell activating antigen is CD3.

In a second object the present invention relates to a pharmaceutical composition comprising a bispecific antibody of the present invention.

In a third object the present invention relates to a bispecific antibody of the present invention for the treatment of cancer. In another embodiment, use of the bispecific antibody as a medicament is provided. Preferably said use is for the treatment of cancer.

In further objects the present invention relates to a nucleic acid sequence comprising a sequence encoding a heavy chain of a bispecific antibody of the present invention, a nucleic acid sequence comprising a sequence encoding a light chain of a bispecific antibody of the present invention, an expression vector comprising a nucleic acid sequence of the present invention and to a prokaryotic or eukaryotic host cell comprising a vector of the present invention. In addition a method of producing an antibody comprising culturing the host cell so that the antibody is produced is provided.

In a further embodiment an immunoconjugate comprising the bispecific antibody of the invention and a cytotoxic agent is provided.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a-e : Schematic illustration of exemplary bispecific antibody formats wherein the two Fab fragments are connected to each other by one linker. a) Fab-Crossfab molecule C-terminal, b) Fab-Crossfab molecule N-terminal c) (Fab)2-Crossfab molecule C-terminal d) (Fab)2-Crossfab molecule N-terminal e) Fab-Crossfab-Fab molecule.

FIGS. 2A-B: Analysis of hu Fab(MCSP)-Crossfab(CD3) production and purification: SDS-Page: 4-12% Bis/Tris (NuPage [invitrogen]; coomassie stained): A 1—Mark 12 (invitrogen), 2—hu Fab(MCSP)-Crossfab(CD3) non reduced; B 1—Mark 12 (invitrogen), 2—hu Fab(MCSP)-Crossfab(CD3) reduced.

FIG. 3: Analysis Fab(MCSP)-Crossfab(CD3) production and purification. Analytical size exclusion chromatography, Chromatogram A280 (Superdex 200 10/300 GL [GE Healthcare]; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample were injected).

FIGS. 4A-B: Analysis of hu Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) production and purification: SDS-Page: 4-12% Bis/Tris (NuPage [invitrogen]; coomassie stained): A 1—Mark 12 (invitrogen), 2—hu Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) non reduced; B 1—Mark 12 (invitrogen), 2—hu Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) reduced.

FIG. 5: Analysis of hu Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) production and purification. Analytical size exclusion chromatography, Chromatogram A280 (Superdex 200 10/300 GL [GE Healthcare]; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 g sample were injected).

FIGS. 6A-B: Analysis of hu Fab(MCSP)-Crossfab(CD3)-Fab(MCSP) production and purification. SDS-Page: 4-12% Bis/Tris (NuPage [invitrogen]; coomassie stained): A 1—Mark 12 (invitrogen), 2—hu Fab(MCSP)-Crossfab(CD3)-Fab(MCSP) non reduced; B 1—Mark 12 (invitrogen), 2—hu Fab(MCSP)-Crossfab(CD3)-Fab(MCSP) reduced.

FIG. 7: Analysis of hu Fab(MCSP)-Crossfab(CD3)-Fab(MCSP) production and purification. Analytical size exclusion chromatography, Chromatogram A280 (Superdex 200 10/300 GL [GE Healthcare]; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 g sample were injected).

FIGS. 8A-B: Analysis of murine Crossfab(CD3)-Fab(MCSP)-Fab(MCSP) production and purification. SDS-Page: 4-12% Bis/Tris (NuPage [invitrogen]; coomassie stained): A 1—Mark 12 (invitrogen), 2—murine Crossfab(CD3)-Fab(MCSP)-Fab(MCSP) non reduced; B 1—Mark 12 (invitrogen), 2—murine Crossfab(CD3)-Fab(MCSP)-Fab(MCSP) reduced.

FIG. 9: Analysis of murine Crossfab(CD3)-Fab(MCSP)-Fab(MCSP) production and purification. Analytical size exclusion chromatography, Chromatogram A280 (Superdex 200 10/300 GL [GE Healthcare]; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample were injected).

FIG. 10: Killing (as measured by LDH release) of MDA-MB-435 tumor cells upon co-culture with human pan T cells (E:T ratio=5:1) and activation for 20 hours by different concentrations of the hu Fab(MCSP)-Crossfab(CD3) (=“Fab-Crossfab”), hu Fab(MCSP)-Crossfab(CD3)-Fab(MCSP) (=“Fab-Crossfab-Fab”), hu Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) (=“(Fab)2-Crossfab”), as well as the (scFv)2 (antiMCSP/anti huCD3e) (=“(scFv)2”) bispecific molecules. The constructs with bivalent MCSP-targeting show comparable cytotoxic activity compared to the “(scFv)2” construct, whereas the “Fab-Crossfab” construct with monovalent MCSP binding is clearly less potent.

FIG. 11: Comparison of the hu Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) (=“(Fab)2-Crossfab”) and the (scFv)2 (antiMCSP/anti huCD3e) (=“(scFv)2”) construct, Depicted is the LDH release from MDA-MB-435 tumor cells upon co-culture with human pan T cells (E/T ratio=5:1), and activation for 21 hours by different concentrations of the bispecific constructs and corresponding IgGs. The “(Fab)2-Crossfab” induces apoptosis in target cells at least comparably good as the (scFv)2 molecule.

FIG. 12: Comparison of the hu Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) (=“(Fab)2-Crossfab”) and the (scFv)2 (antiMCSP/anti huCD3e) (=“(scFv)2”) construct. Depicted is the LDH release from MV-3 human melanoma tumor cells upon co-culture with human PBMCs (E/T ratio=10:1), and activation for 26 hours by different concentrations of the bispecific constructs and corresponding IgGs. The “(Fab)2-Crossfab” induces apoptosis in target cells at least comparably good as the (scFv)2 molecule.

FIG. 13: LDH release from B16/F10-huMCSP Fluc2, clone 48 tumor cells, induced by primary murine T cell activation with the murine Crossfab(CD3)-Fab(MCSP)-Fab(MCSP) construct (=(Fab)2-CrossFab), targeting human MCSP, as well as the murine CD3. The effector to target cell ratio was 5:1. The assay was analyzed after incubation for 23.5 hours at 37° C., 5% CO2. The construct induces concentration-dependent, T cell-mediated apoptosis of human MCSP-expressing target cells.

FIG. 14: LDH release from B16/F10-huMCSP Fluc2, clone 48 tumor cells, induced by primary murine T cell activation with 50 nM of the murine Crossfab(CD3)-Fab(MCSP)-Fab(MCSP) construct (=(Fab)2-CrossFab), targeting human MCSP, as well as the murine CD3. The effector to target cell ratio was 5:1. The assay was analyzed after incubation for 23.5 hours at 37° C., 5% CO2. The construct induces T cell-mediated apoptosis of human MCSP-expressing target cells. There is only weak hyperactivation of T cells at this concentration of the construct.

FIGS. 15a : Different cytokine levels measured in the supernatant of whole blood after treatment with 1 nM of different CD3-MCSP bispecific constructs (hu Fab(MCSP)-Fab(MCSP)-Crossfab(CD3) (=“(Fab)2-Crossfab”) and (scFv)2 (antiMCSP/anti huCD3e) (=“(scFv)2”)) in the presence (a,b) or absence (c,d) of Colo-38 tumor cells for 24 hours. 280 μl whole blood were plated per well of a 96-well plate and 30 000 Colo-38 cells added, as indicated. The main cytokine that was secreted upon activation of T cells in the presence of Colo-38 tumor cells, is IL-6, followed by IFNgamma. In addition, also the levels of granzyme B increased enormously upon activation of T cells in the presence of target cells. In general, the “(scFv)2” construct elevated the levels of TNF and IFNgamma, as well as granzyme B in the presence of target cells (a and b) a bit more compared to the other bispecific construct.

There was no significant secretion of Th2 cytokines (IL-10 and IL-4) upon activation of T cells by the bispecific constructs in the presence (or absence) of target cells. In this assay there was also a weak secretion of IFNgamma, induced by the “(Fab)2-Crossfab” construct in the absence of target cells.

FIG. 16: Surface expression level of the late activation marker CD25 on murine pan T cells, isolated from splenocytes. Murine pan T cells were incubated with 50 nM of the murine Crossfab(CD3)-Fab(MCSP)-Fab(MCSP) construct (=(Fab)2-CrossFab) bispecific construct (targeting murine CD3, as well as human MCSP), in the presence or absence of B16/F10-huMCSP Fluc2 clone 48 tumor target cells, as indicated (E:T ratio is 10:1). Depicted is the expression level of the late activation marker CD25 on CD8+ T cells after 70 hours. Up-egulation of CD25 on CD8+ T cells with the (Fab)2-CrossFab construct occurs only in the presence of target cells. The reference IgGs, used adjusted to the same molarity, were not able to up-regulate CD25.

FIGS. 17A-B: Analysis of Fab(CD33)-CrossFab (CD3) production and purification. SDS-Page: A 3-8% Tris/Acetate (NuPage [invitrogen]; coomassie stained): B 1—HiMark (invitrogen), 2—Fab(CD33)-CrossFab (CD3) non reduced; b) 4-12% Bis/Tris (NuPage [invitrogen]: 1—Mark 12 (invitrogen), 2—Fab(CD33)-CrossFab (CD3) reduced.

FIG. 18: Analysis of Fab(CD33)-CrossFab (CD3) production and purification. Analytical size exclusion chromatography, Chromatogram A280 (Superdex 200 10/300 GL [GE Healthcare]; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample were injected).

FIG. 19: Killing (as measured by LDH release) of MV-3 tumor cells upon co-culture with human PBMCs (E:T ratio=10:1) and activation for 24 hours by different concentrations of CD3-MCSP bispecific constructs (hu Fab(MCSP)-Crossfab(CD3); designated as “1+1 non-Fc”, and the (scFv)2 (antiMCSP/anti huCD3e) (=“(scFv)2”) reference molecule). The “1+1 non-Fc” construct induces apoptosis in MV-3 target cells with a calculated EC50 of 25.4 μM, whereas the calculated EC50 for the “(scFv)2” reference molecule is 57 μM, showing a slight better potency of the “1+1 non-Fc” molecule in terms of EC50.

FIGS. 20a-b : Activation of CD4+ or CD8+ T cells, as measured by up-regulation of CD69 ( a), respective increase of CD69-positive cells (b) in the presence of huMCSP-positive MV-3 tumor cells upon co-culture with human PBMCs (E:T ratio=10:1), treated with the CD3-MCSP bispecific constructs (hu Fab(MCSP)-Crossfab(CD3); designated as “1+1 non-Fc”, and the (scFv)2 (antiMCSP/anti huCD3e) (=“(scFv)2”) reference molecule, respectively) for ˜24 hours. In general, the CD69 median values are higher on CD8+ T cells compared to CD4+T cells. There is a clear concentration-dependent increase in both, CD69 median values, as well percentage of CD69 positive cells for both constructs.

FIG. 21: Illustration of (scFv)2 reference molecule.

FIG. 22: Analysis of (scFv)2 (antiMCSP/anti huCD3e) production and purification. SDS-Page: 4-12% Bis/Tris (NuPage [invitrogen]; coomassie stained): 1—Mark 12 (invitrogen), 2—(scFv)2 (antiMCSP/anti huCD3e) reduced; 3—(scFv)2 (antiMCSP/anti huCD3e) non reduced

FIG. 23: Analysis of (scFv)2 (antiMCSP/anti huCD3e) production and purification Analytical size exclusion chromatography, Chromatogram A280 (Superdex 75 10/300 GL [GE Healthcare]; 2 mM MOPS pH 7.3, 150 mM NaCl, 0.02% (w/v) NaCl; 50 μg sample ((scFv)2 (antiMCSP/anti huCD3e)) were injected).

FIGS. 24a-e : Schematic illustration of exemplary bispecific antibody formats wherein the two Fab fragments are connected to each other by two linkers a) Fab=Crossfab molecule C-terminal, b) Fab=Crossfab molecule N-terminal c) Fab-Fab=Crossfab molecule and Fab=Fab=Crossfab molecule C-terminal d) Fab-Fab=Crossfab molecule and Fab=Fab=Crossfab molecule N-terminal e) Fab-Crossfab=Fab and Fab=Crossfab=Fab molecule.

FIG. 25: Analysis of hu Fab(MCSP)=Crossfab(CD3) production and purification: SDS-Page: 4-12% Bis/Tris (NuPage [invitrogen]; coomassie stained): A) hu Fab(MCSP)=Crossfab(CD3) reduced; B) Mark 12 (invitrogen), C) hu Fab(MCSP)=Crossfab(CD3) non reduced;

FIG. 26: Killing (as measured by LDH release) of MCSP-positive MV-3 human melanoma cells upon co-culture with human PBMCs (E:T ratio=10:1), treated with different CD3-MCSP bispecific constructs (hu Fab(MCSP)=Crossfab (CD3), designated as “1+1 non-Fc, linked LC”; hu Fab(MCSP)-Crossfab(CD3), designated as “1+1 non-Fc”; and the (scFv)2 (antiMCSP/anti huCD3e) (=“(scFv)2”) reference molecule, respectively) for ˜24 hours. Human PBMCs were isolated from fresh blood of healthy volunteers. EC50 values were determined using Graph Pad Prism software.

FIGS. 27a-b : Surface expression level of the early activation marker CD69 on human CD4and CD8 T cells after 24 hours incubation with 10 nM, 80 pM or 16 pM of the non-Fc T cell bispecific constructs targeting human CD3 and human MCSP (hu Fab(MCSP)=Crossfab(CD3), designated as “1+1 non-Fc, linked LC”; hu Fab(MCSP)-Crossfab(CD3), designated as “1+1 non-Fc”; and the (scFv)2(antiMCSP/anti huCD3e) (=“(scFv)2”) reference molecule, respectively)) in the presence of human MCSP-expressing MV-3 melanoma target cells (E:T ratio=10:1).

(a) Shows the median expression values of CD69, (b) depicts the percentage of CD69 positive T cells.

FIG. 28: Thermal stability of Fab-Crossfab molecule with two linkers (antiMCSP/anti huCD3) in comparison to the “(scFv)2” reference molecule (antiMCSP/anti huCD3). Dynamic Light Scattering. measured in a temperature ramp from 25-75° C. at 0.05° C./min. Fab-Crossfab molecule with two linkers is shown in black and the “(scFv)2” reference molecule is shown in grey.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

I. Definitions

“Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

The term “hypervariable region” or “HVR,” as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the “complementarity determining regions” (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).) The terms hypervariable regions (HVRs) and complementarity determining regions (CDRs), are used herein interchangeably in reference to portions of the variable region that form the antigen binding regions. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

TABLE 1 CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-32 26-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102 V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR3 89-97 91-96 89-97 ¹Numbering of all CDR definitions in Table 1 is according to the numbering conventions set forth by Kabat et al. (see below). ²“AbM” with a lowercase “b” as used in Table 1 refers to the CDRs as defined by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable region sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody variable region are according to the Kabat numbering system.

With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise “specificity determining residues,” or “SDRs,” which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. In particular the term “antibody” also includes the bispecific antibodies of the invention comprising at least two Fab fragments connected to each other by two linkers and which are devoid of a Fc domain.

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.

A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of “humanized antibodies” encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.

The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other preferred forms of “chimeric antibodies” encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to C1q binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as “class-switched antibodies.”. Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See e.g. Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab′, Fab′-SH, F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. scFv antibodies are, e.g. described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96). In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full length antibodies.

As used herein, “Fab fragment” refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CH1) of a heavy chain. The bispecific antibodies of the invention comprise at least two Fab fragments, wherein either the variable regions or the constant regions of the heavy and light chain of the second Fab fragment are exchanged. Due to the exchange of either the variable regions or the constant regions, said second Fab fragment is also referred to as “cross-Fab fragment” or “xFab fragment” or “crossover Fab fragment”. Two different chain compositions of a crossover Fab molecule are possible and comprised in the bispecific antibodies of the invention: On the one hand, the variable regions of the Fab heavy and light chain are exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1), and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). This crossover Fab molecule is also referred to as CrossFab_((VLVH)). On the other hand, when the constant regions of the Fab heavy and light chain are exchanged, the crossover Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1). This crossover Fab molecule is also referred to as CrossFab_((CLCH1).)

The Fab fragments are connected to each other by at least two peptide linkers, as outlined below.

If not otherwise defined, “connected” means that the Fab fragments are linked by peptide bonds, either directly or via one or more peptide linker The term “peptide linker” as used within the invention denotes a peptide with amino acid sequences, which is preferably of synthetic origin. These peptide linkers according to invention are used to connect one of the Fab fragments to the C-or N-terminus of the other Fab fragment to form a multispecific antibody according to the invention. Preferably said peptide linkers are peptides with an amino acid sequence with a length of at least 5 amino acids, preferably with a length of 5 to 100, more preferably of 10 to 50 amino acids. In one embodiment said peptide linker is (GxS)n or (GxS)nGm with G =glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4,n=2, 3, 4 or 5 and m=0, 1, 2 or 3) (SEQ ID NOS 204, 206, 205 and 207, respectively, in order of appearance), preferably x=4 and n=2 or 3, more preferably with x=4, n=2. Additionally, linkers may comprise (a portion of) an immunoglobulin hinge region. In one embodiment said peptide linker is (G₄S)₂ (SEQ ID: NO 28). Other peptide linkers suitable for connecting the Fab fragments, for example, (G₄S)₆-GG (SEQ ID NO: 147) or (SG₃)₂-(SEG₃)₄-(SG₃)-SG (SEQ ID NO: 148), or EPKSC(D)-(G₄S)₂ (SEQ ID NOs 145 and 146).

The term “antigen binding domain” refers to the part of an antigen binding molecule that comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antigen binding molecule may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “antigen-binding site of an antibody” when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding. The antigen-binding portion of an antibody comprises amino acid residues from the “complementary determining regions” or “CDRs”. “Framework” or “FR” regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chain variable domains of an antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Especially, CDR3 of the heavy chain is the region which contributes most to antigen binding and defines the antibody's properties. CDR and FR regions are determined according to the standard definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or those residues from a “hypervariable loop”.

The term “epitope” includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody.

The term “Fc domain” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. For example in natural antibodies, the Fc domain is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains in IgG, IgA and IgD isotypes; IgM and IgE Fc domains contain three heavy chain constant domains (C_(H) domains 2-4) in each polypeptide chain. The bispecific antibodies of the invention are devoid of the Fc domain. “Devoid of the Fc domain” as used herein means that the bispecific antibodies of the invention do not comprise a CH2, CH3 or CH4 domain; i.e. the constant heavy chain consists solely of one or more CH1 domains.

“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

As used herein, the term “binding” or “specifically binding” means that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed on a BIACORE® instrument (GE Healthcare, Marlborough, Mass) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002). In one embodiment, the extent of binding of an antigen binding moiety to an unrelated protein is less than about 10% of the binding of the antigen binding moiety to the antigen as measured, e.g., by SPR. In certain embodiments, an antigen binding moiety that binds to the antigen, or an antigen binding molecule comprising that antigen binding moiety, has a dissociation constant (K_(D)) of <1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M).

An “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

In one embodiment, the extent of binding of a bispecific antibody that specifically binds a first antigen and asecond antigen to an unrelated, protein is less than about 10% of the binding of the antibody to the first or second antigen as measured, e.g., by a radioimmunoassay (RIA) or flow cytometry (FACS). In certain embodiments, a bispecific antibody that specifically binds a first antigen and a second antigen has a dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certain embodiments, a bispecific antibody that specifically binds a first antigen and a second antigen binds to an epitope of the first antigen or the second antigen that is conserved among the first or second antigen from different species.

In one embodiment, the extent of binding of a bispecific antibody that specifically binds to a T-cell activating antigen and a Tumor Antigen (TA) to an unrelated protein is less than about 10% of the binding of the antibody to a T-cell activating antigen or a Tumor Antigen (TA) as measured, e.g., by a radioimmunoassay (RIA) or flow cytometry (FACS). In certain embodiments, a bispecific antibody that specifically binds T-cell activating antigen and a Tumor Antigen (TA) has a dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certain embodiments, a bispecific antibody that specifically binds a T-cell activating antigen and a Tumor Antigen (TA) binds to an epitope of a T-cell activating antigen or a Tumor Antigen (TA) that is conserved among a T-cell activating antigen or a Tumor Antigen (TA) from different species.

The terms “A bispecific antibody that specifically binds a T cell activating antigen and a Tumor Antigen (TA)” refers to a bispecific antibody that is capable of binding a T cell activating antigen and a Tumor Antigen with sufficient affinity such that the antibody is useful in mediating a T-cell mediated immune response in or near cells expressing a Tumor Antigen. In a particular embodiment the T cell activating antigen is the CD3 T-Cell Co-Receptor (CD3) antigen, particularly human or cynomolgus CD3, most particularly human CD3. In some embodiments, the T cell activating antigen is the epsilon subunit of CD3. In other embodiments, the T cell activating antigen is the alpha or beta subunit of CD3.

In one embodiment, the bispecific antibody that specifically binds a T cell activating antigen and a Tumor Antigen (TA) can compete with monoclonal antibody H2C (described in PCT publication no. WO2008/119567) for binding an epitope of CD3. In another embodiment, the bispecific antibody that specifically binds a T cell activating antigen and a Tumor Antigen (TA) can compete with monoclonal antibody V9 (described in Rodrigues et al., Int J Cancer Suppl 7, 45-50 (1992) and U.S. Pat. No. 6,054,297) for binding an epitope of CD3. In yet another embodiment, the bispecific antibody that specifically binds a T cell activating antigen and a Tumor Antigen (TA) can compete with monoclonal antibody FN18 (described in Nooij et al., Eur J Immunol 19, 981-984 (1986)) for binding an epitope of CD3. In one embodiment the bispecific antibody comprises an antigen binding moiety that can compete with monoclonal antibody CH2527 (Sequence ID 157 and 158) or an affinity matured variant thereof for binding to an epitope of CD3.

An “activating T cell antigen” as used herein refers to an antigenic determinant expressed on the surface of a T lymphocyte, particularly a cytotoxic T lymphocyte, which is capable of inducing T cell activation upon interaction with an antigen binding molecule. Specifically, interaction of an antigen binding molecule with an activating T cell antigen may induce T cell activation by triggering the signaling cascade of the T cell receptor complex. In a particular embodiment the activating T cell antigen is CD3.

“T cell activation” as used herein refers to one or more cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. The T cell activating bispecific antigen binding molecules of the invention are capable of inducing T cell activation. Suitable assays to measure T cell activation are known in the art described herein.

The term “CD3 T-Cell Co-Receptor (CD3)”, as used herein, refers to a protein complex and is composed of four distinct chains. In mammals, the complex contains a CD3γ chain, a CD3δ chain, and two CD3∈ chains. These chains associate with a molecule known as the T cell receptor (TCR) and the ζ-chain to generate an activation signal in T lymphocytes. The term “CD3 T-Cell Co-Receptor (CD3)” includes any native CD3 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated, preferably from a human source. The term encompasses “full-length,” unprocessed CD3 as well as any form of CD3 that results from processing in the cell. The term also encompasses naturally occurring variants of CD3, e.g., splice variants or allelic variants. In a preferred embodiment, the term CD3 T-Cell Co-Receptor refers to human or cynomolgus CD3, particularly human CD3. In some embodiments, the T cell activating antigen is the epsilon subunit of CD3. In other embodiments, the T cell activating antigen is the alpha or beta subunit of CD3. An exemplary sequence of human CD3 is given in SEQ ID NO.: 103.

The term “Tumor Antigen (TA)”, as used herein, refers to tumor-associated antigens as well as tumor-specific antigens, i.e. any immunogenic epitope (e.g., protein) expressed by a tumor cell. The protein may be expressed by non tumor cells but be immunogenic only when expressed by a tumor cell. Alternatively, the protein may be expressed by tumor cells, but not normal cells. Preferably, an anti-TA antibody of the invention binds to the extracellular domain of TA. In one preferred embodiment said Tumor Antigen is a human Tumor Antigen. Exemplary Tumor Antigens include but are not limited to Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSPB, UniProt Q6UVK1, NCBI Accession NP_001888), Fibroblast Activation Protein (FAP, Uni Prot Q12884, Q86Z29, Q99998; NCBI Accession NP_004451), Epidermal Growth Factor Receptor (EGFR, also known as ErbB1 and Her1, UniProt P00533; NCBI Accession NP_958439, NP_958440), Carcinoembryonic Antigen (CEA, also known as Carcinoembryonic antigen-related cell adhesion molecule 5 or CD66e; UniProt P06731, NCBI Accession NP_004354) and CD33 (also known as gp76 or Sialic acid-binding Ig-like lectin 3 (Siglec-3), UniProt P20138, NCBI Accession NP_001076087, NP_001171079).

In one embodiment the bispecific antibody of the invention comprises at least one antigen binding site that is specific for Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP).

Antibody specificity refers to selective recognition of the antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. “Bispecific antibodies” according to the invention are antibodies which have two different antigen-binding specificities. Antibodies of the present invention are specific for two different antigens, i.e. for a first antigen and a second antigen. Provided herein is a bispecific antibody, with binding specificities for a Tumor Antigen (TA) and a T-cell activating antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of TA. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express TA.

The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.

The term “bispecific” antibody as used herein denotes an antibody that has at least two binding sites each of which bind to different epitopes of the same antigen or a different antigen.

The antibody provided herein is a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. Provided herein is a bispecific antibody, with binding specificities for a first antigen and a second antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of a first antigen or a second antigen.

Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a first antigen or a second antigen.

In one embodiment, the extent of binding of a bispecific antibody that specifically binds to a T-cell activating antigen and a Tumor Antigen (TA) to an unrelated protein is less than about 10% of the binding of the antibody to a T-cell activating antigen or a Tumor Antigen (TA) as measured, e.g., by a radioimmunoassay (RIA) or flow cytometry (FACS). In certain embodiments, a bispecific antibody that specifically binds T-cell activating antigen and a Tumor Antigen (TA) has a dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certain embodiments, a bispecific antibody that specifically binds a T-cell activating antigen and a Tumor Antigen (TA) binds to an epitope of a T-cell activating antigen or a Tumor Antigen (TA) that is conserved among a T-cell activating antigen or a Tumor Antigen (TA) from different species.

The term “valent” as used within the current application denotes the presence of a specified number of binding sites in an antibody molecule. As such, the terms “bivalent”, “tetravalent”, and “hexavalent” denote the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antibody molecule. The bispecific antibodies according to the invention are at least “bivalent” and may be “trivalent” or “multivalent” (e.g. “tetravalent” or “hexavalent”).

Antibodies of the present invention have two or more binding sites and are bispecific. That is, the antibodies may be bispecific even in cases where there are more than two binding sites (i.e. that the antibody is trivalent or multivalent).

An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein.

“No substantial cross-reactivity” means that a molecule (e.g., an antibody) does not recognize or specifically bind an antigen different from the actual target antigen of the molecule (e.g. an antigen closely related to the target antigen), particularly when compared to that target antigen. For example, an antibody may bind less than about 10% to less than about 5% to an antigen different from the actual target antigen, or may bind said antigen different from the actual target antigen at an amount selected from the group consisting of less than about 10%, 9%, 8% 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%, preferably less than about 2%, 1%, or 0.5%, and most preferably less than about 0.2% or 0.1% antigen different from the actual target antigen.

“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

“Isolated nucleic acid encoding a bispecific antibody of the invention” refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

“Isolated nucleic acid encoding a bispecific antibody that specifically binds a T-Cell activating antigen and a Tumor Antigen (TA)” refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

The term “amino acid” as used within this application denotes the group of naturally occurring carboxy α-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

As used herein, the expressions “cell”, “cell line”, and “cell culture” are used interchangeably and all such designations include progeny. Thus, the words “transfectants” and “transfected cells” include the primary subject cell and cultures derived there from without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.

The term “N-terminus” denotes the last amino acid of the N-terminus, the term “C-terminus” denotes the last amino acid of the C-terminus.

The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

An “effective amount” of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

The term “cancer” as used herein refers to proliferative diseases, such as lymphomas, lymphocytic leukemias, lung cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma and Ewings sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers.

The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

II. Compositions and Methods

A. Exemplary Bispecific Antibodies

The present invention relates to bispecific antibodies comprising at least two Fab fragments, wherein the first Fab fragment comprises at least one antigen binding site specific for a first antigen; and the second Fab fragment comprises at least one antigen binding site specific for a second antigen, wherein either the variable regions or the constant regions of the heavy and light chain of the second Fab fragment are exchanged; and wherein the two Fab fragments are connected to each other by two peptide linkers; and wherein the bispecific antibody is devoid of a Fc domain. Preferably said peptide linker is a peptide with an amino acid sequence with a length of at least 5 amino acids, preferably with a length of 5 to 100, more preferably of 10 to 50 amino acids. In one embodiment said peptide linker is (GxS)n or (GxS)nGm with G =glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3) (SEQ ID NOS 204, 206, 205 and 207, respectively, in order of appearance), preferably x=4 and n=2 or 3, more preferably with x=4, n=2. In one embodiment said peptide linker is (G₄S)₂ (SEQ ID NO: 28). The peptide linkers are used to connect the first and the second Fab fragment.

In one embodiment the first Fab fragment is connected to the N-terminus of the second Fab fragment by two peptide linkers. Depending on whether the variable or the constant domains of the heavy and the light chains of the second Fab fragment are exchanged, different bispecific antibody molecules are possible when the first Fab fragment is connected to the N-terminus of the second Fab fragment by two peptide linkers.

In one embodiment the variable domains of the second Fab fragment are exchanged (i.e. the second Fab fragment is a CrossFab_((VHVL))), and the C-terminus of the heavy chain of the first Fab fragment is connected to the N-terminus of the VLCH1 chain of the second Fab fragment by a peptide linker, and the C-terminus of the light chain of the first Fab fragment is connected to the N-terminus of the VHCL chain of the second Fab fragment by a peptide linker. Thus, in one embodiment the bispecific antibody comprises two chains: a light chain (VLCL) of the first Fab fragment connected to the VHCL chain of the second Fab fragment via a peptide linker (VLCL-linker-VHCL) and the heavy chain (VHCH1) of the first Fab fragment connected to the VLCH1 chain of the second Fab fragment via a peptide linker (VHCH1-linker-VLCH1).

In another embodiment the constant domains of the second Fab fragment are exchanged (i.e the second Fab fragment is a CrossFab_((CLCH1))) and the C-terminus of the heavy chain of the first Fab fragment is connected to the N-terminus of the VHCL chain of the second Fab fragment by a peptide linker, and the light chain of the first Fab fragment is connected to the N-terminus of the VLCH1 chain by a peptide linker. Thus, in one embodiment the bispecific antibody comprises two chains: a light chain (VLCL) of the first Fab fragment connected to the VLCH1 chain of the second Fab fragment via a peptide linker (VLCL-linker-VLCH1), and the heavy chain (VHCH1) of the first Fab fragment connected to the VHCL chain of the second Fab fragment via a peptide linker (VHCH1-linker-VHCL).

In one embodiment the first Fab fragment is connected to the C-terminus of the second Fab fragment by two peptide linkers. Depending on whether the variable or the constant domains of the heavy and the light chains of the second Fab fragment are exchanged different bispecific antibody molecules are possible when the first Fab fragment is connected to the C-terminus of the second Fab fragment by two peptide linkers.

In one embodiment the variable domains of the second Fab fragment are exchanged (i.e. the second Fab fragment is a CrossFab_((VHVL))), and the N-terminus of the heavy chain of the first Fab fragment is connected to the C-terminus of the VLCH1 chain of the second Fab fragment by a peptide linker, and the N-terminus of the light chain of the first Fab fragment is connected to the C-terminus of the VHCL chain of the second Fab fragment by a peptide linker. Thus, in one embodiment the bispecific antibody comprises two chains: the VHCL chain of the second Fab fragment connected to a light chain (VLCL) of the first Fab fragment via a peptide linker (VHCL-linker-VLCL) and the VLCH1 chain of the second Fab fragment connected to the heavy chain (VHCH1) of the first Fab fragment via a peptide linker (VLCH1-linker-VHCH1).

In another embodiment the constant domains of the second Fab fragment are exchanged (i.e the second Fab fragment is a CrossFab (CLCH1)) and the N-terminus of the heavy chain of the first Fab fragment is connected to the C-terminus of the VHCL chain of the second Fab fragment by a peptide linker, and the N-terminus of the light chain of the first Fab fragment is connected to the C-terminus of the VLCH1 chain by a peptide linker. Thus, in one embodiment the bispecific antibody comprises two chains: the VLCH1 chain of the second Fab fragment connected to a light chain (VLCL) of the first Fab fragment via a peptide linker (VLCH1-linker-VLCL), and the VHCL chain of the second Fab fragment connected to the heavy chain (VHCH1) of the first Fab fragment via a peptide linker (VHCL-linker-VHCH1). The bispecific antibodies according to the invention are at least bivalent and can be trivalent or multivalent e.g. tetravalent. In one embodiment said bispecific antibodies are bivalent (1+1 format) with one binding site each targeting a first antigen and a second antigen, respectively. In another embodiment said bispecific antibodies are trivalent (2+1 format) with two binding sites each targeting a first antigen and one binding site targeting a second antigen, as detailed in the following section.

In one embodiment said antibody additionally comprises a third Fab fragment. In another embodiment said third Fab fragment comprises at least one antigen binding site specific for the first or second antigen, preferably for the first antigen.

In one embodiment the third Fab fragment is connected to the N or C-terminus of the first Fab fragment. In one embodiment the third Fab fragment is connected to the first Fab fragment via at least one peptide linker. Preferably said peptide linker is a (G4S)2 linker (SEQ ID NO: 28).

In one embodiment the third Fab fragment is connected to the N or C-terminus of the light chain or the heavy chain of the first Fab fragment. Depending on which terminus of the first Fab fragment is connected to the second Fab fragment (as detailed above), the third Fab fragment is connected on the opposite (free) terminus of the first fragment. The third Fab fragment can be connected by either one or two linkers.

In one embodiment, the bispecific antibody of the invention comprises three Fab fragments wherein said Fab fragments and said linkers are connected in the following order from N-terminal to C-terminal direction: Fab fragment 3- (1 or 2 linkers)-Fab fragment 1- (2 linkers)-Fab fragment 2, wherein either the variable regions or the constant regions of the heavy and light chain of the second Fab fragment are exchanged. In this embodiment the C-terminus of the third Fab fragment is connected to the N-terminus of the first Fab fragment. As detailed above, the Fab fragments can be connected to each other via the heavy or the light chains. In one embodiment the C-terminus of the heavy chain of the third Fab fragment is connected to the N-terminus of the heavy chain of the first Fab fragment via a peptide linker; and the first Fab fragment is connected to the second Fab fragment by two linkers, wherein either the variable regions or the constant regions of the heavy and light chain of the second Fab fragment are exchanged. Depending on whether the variable or the constant domains of the heavy and the light chains of the second Fab fragment are exchanged different bispecific antibody molecules are possible.

In one embodiment the variable domains of the second Fab fragment are exchanged (i.e. the second Fab fragment is a CrossFab_((VHVL))), and the chains of the three Fab fragments are connected in the following order from N-terminal to C-terminal direction: VHCH1-linker-VHCH1-linker-VLCH1. In one embodiment the bispecific antibody comprises three chains: a light chain (VLCL) of the third Fab fragment, a light chain (VLCL) of the first Fab fragment connected to the VHCL chain of the second Fab fragment by a peptide linker (VLCL-linker-VHCL), and the heavy chain of the third fragment connected to the heavy chain of the first Fab fragment which itself is connected to the VLCH1 chain of the second Fab fragment via a peptide linker (VHCH1-linker-VHCH1-linker-VLCH1). In another embodiment said third Fab fragment is connected to the first Fab fragment by two linkers, and the bispecific antibody comprises two chains: the light chain (VLCL) of the third Fab fragment connected by linker to a light chain (VLCL) of the first Fab fragment which is in turn connected to the VHCL chain of the second Fab fragment by a peptide linker (VLCL-linker-VLCL-linker-VHCL), and the heavy chain of the third fragment connected to the heavy chain of the first Fab fragment which itself is connected to the VLCH1 chain of the second Fab fragment via a peptide linker (VHCH1-linker-VHCH1-linker-VLCH1).

In one embodiment the constant domains of the second Fab fragment are exchanged (i.e. the second Fab fragment is a CrossFab_((CLCH1))), and the chains of the three Fab fragments are connected in the following order from N-terminal to C-terminal direction: VHCH1-linker-VHCH1-linker-VHCL. In one embodiment the bispecific antibody comprises three chains: a light chain (VLCL) of the third Fab fragment, a light chain (VLCL) of the first Fab fragment to the VLCH1 chain of the second Fab fragment by a peptide linker (VLCL-linker-VLCH1), and the heavy chain of the third fragment connected to the heavy chain of the first Fab fragment which itself is connected to the VHCL chain of the second Fab fragment via a peptide linker (VHCH1-linker-VHCH1-linker-VHCL). In another embodiment said third Fab fragment is connected to the first Fab fragment by two linkers, and the bispecific antibody comprises two chains: a light chain (VLCL) of the third Fab fragment connected to a light chain (VLCL) of the first Fab fragment which is in turn connected to the VLCH1 chain of the second Fab fragment by a peptide linker (VLCL-linker-VLCL-linker-VLCH1), and the heavy chain of the third fragment connected to the heavy chain of the first Fab fragment which itself is connected to the VHCL chain of the second Fab fragment via a peptide linker (VHCH1-linker-VHCH1-linker-VHCL)

In one embodiment the bispecific antibody of the invention comprises three Fab fragments wherein said Fab fragments and said linker are connected in the following order from N-terminal to C-terminal direction: Fab fragment 2- (2 linkers)-Fab fragment 1- (1 or 2 linkers)-Fab fragment 3, wherein either the variable regions or the constant regions of the heavy and light chain of the second Fab fragment are exchanged. In this embodiment the N-terminus of the third Fab fragment is connected to the C-terminus of the first Fab fragment. As detailed above, the Fab fragments can be connected to each other via the heavy or the light chains. In one embodiment the N-terminus of the heavy chain of the third Fab fragment is connected to the C-terminus of the heavy chain of the first Fab fragment via a peptide linker; and the first Fab fragment is connected to the second Fab fragment by two linkers, wherein either the variable regions or the constant regions of the heavy and light chain of the second Fab fragment are exchanged. Depending on whether the variable or the constant domains of the heavy and the light chains of the second Fab fragment are exchanged different bispecific antibody molecules are possible.

In one embodiment the variable domains of the second Fab fragment are exchanged (i.e. the second Fab fragment is a CrossFab_((VHVL))), and the chains of the three Fab fragments are connected in the following order from N-terminal to C-terminal direction: VLCH1-linker-VHCH1-linker-VHCH1. In one embodiment the bispecific antibody comprises three chains: a light chain (VLCL) of the third Fab fragment, a VHCL chain of the second Fab fragment connected to a light chain (VLCL) of the first Fab fragment by a peptide linker (VHCL-linker-VLCL), and the VLCH1 chain of the second Fab fragment connected to the heavy chain of the first fragment which itself is connected to the heavy chain of the first Fab fragment via a peptide linker (VLCH1-linker-VHCH1-linker-VHCH1). In another embodiment said third Fab fragment is connected to the first Fab fragment by two linkers, and the bispecific antibody comprises two chains: a VHCL chain of the second Fab fragment connected to a light chain (VLCL) of the first Fab fragment by a peptide linker, which is in turn connected to a light chain (VLCL) of the third Fab fragment by a peptide linker (VHCL-linker-VLCL-linker-VLCL), and the VLCH1 chain of the second Fab fragment connected to the heavy chain of the first fragment which itself is connected to the heavy chain of the first Fab fragment via a peptide linker (VLCH1-linker-VHCH1-linker-VHCH1).

In one embodiment the constant domains of the second Fab fragment are exchanged (i.e. the second Fab fragment is a CrossFab_((CLCH1))), and the chains of the three Fab fragments are connected in the following order from N-terminal to C-terminal direction: VHCL-linker-VHCH1-linker-VHCH1. In one embodiment the bispecific antibody comprises three chains: a light chain (VLCL) of the third Fab fragment, and a VLCH1 chain of the second Fab fragment connected to a light chain (VLCL) of the first Fab fragment (VLCH1-linker-VLCL), and the VHCL chain of the second Fab fragment connected to the heavy chain of the first fragment which itself is connected to the heavy chain of the first Fab fragment via a peptide linker (VHCL-linker-VHCH1-linker-VHCH1). In another embodiment said third Fab fragment is connected to the first Fab fragment by two linkers, and the bispecific antibody comprises two chains: a VLCH1 chain of the second Fab fragment connected to a light chain (VLCL) of the first Fab fragment, which is in turn connected to a light chain of the third fragment (VLCH1-linker-VLCL-linker-VLCL) and the VHCL chain of the second Fab fragment connected to the heavy chain of the first fragment which itself is connected to the heavy chain of the first Fab fragment via a peptide linker (VHCL-linker-VHCH1-linker-VHCH1).

In another embodiment the third Fab fragment is connected to N or C-terminus of the light chain or the heavy chain of the second Fab fragment. In one embodiment the third Fab fragment is connected to the second Fab fragment via a peptide linker. Preferably said peptide linker is a (G4S)2 linker (SEQ ID NO: 28). As detailed above, the Fab fragments can be connected to each other via the heavy or the light chains.

In one embodiment the bispecific antibody of the invention comprises three Fab fragments wherein said Fab fragments and said linker are connected in the following order from N-terminal to C-terminal direction: Fab fragment 1- (2 linkers)-Fab fragment 2- (1 or 2 linkers)-Fab fragment 3, wherein either the variable regions or the constant regions of the heavy and light chain of the second Fab fragment are exchanged. In one embodiment the N-terminus of the third Fab fragment is connected to the C-terminus of the second Fab fragment.

In another embodiment the C-terminus of the heavy chain of the third Fab fragment is connected to the N-terminus of the second Fab fragment via a peptide linker; and the first Fab fragment is connected to the second Fab fragment by two linkers, wherein either the variable regions or the constant regions of the heavy and light chain of the second Fab fragment are exchanged.

Depending on whether the variable or the constant domains of the heavy and the light chains of the second Fab fragment are exchanged different bispecific antibody molecules are possible.

In one embodiment the variable domains of the second Fab fragment are exchanged (i.e. the second Fab fragment is a CrossFab_((VHVL))), and the chains of the three Fab fragments are connected in the following order from N-terminal to C-terminal direction:VHCH1-linker-VLCH1-linker-VHCH1. In one embodiment the bispecific antibody comprises three chains: a light chain (VLCL) of the third Fab fragment, a light chain (VLCL) of the first Fab fragment connected to a VHCL chain of the second Fab fragment by a linker (VLCL-linker-VHCL), and the heavy chain of the third fragment connected to the N-terminus of the VLCH1 chain of the second Fab fragment, and the C-terminus of said VLCH1 chain connected to the N-terminus of the heavy chain of the first Fab fragment via a peptide linker (VHCH1-linker-VLCH1-linker-VHCH1). In another embodiment said third Fab fragment is connected to the second Fab fragment by two linkers, and the bispecific antibody comprises two chains: a light chain (VLCL) of the first Fab fragment connected to a VHCL chain of the second Fab fragment by a linker, which is in turn connected to the light chain of the third Fab fragment (VLCL-linker-VHCL-linker-VLCL), and the heavy chain of the third fragment connected to the N-terminus of the VLCH1 chain of the second Fab fragment, and the C-terminus of said VLCH1 chain connected to the N-terminus of the heavy chain of the first Fab fragment via a peptide linker (VHCH1-linker-VLCH1-linker-VHCH1).

In one embodiment the constant domains of the second Fab fragment are exchanged (i.e. the second Fab fragment is a CrossFab_((CLCH1))), and the chains of the three Fab fragments are connected in the following order from N-terminal to C-terminal direction:VHCH1-linker-VHCL-linker-VHCH1. In one embodiment the bispecific antibody comprises three chains: a light chain (VLCL) of the third Fab fragment, a light chain (VLCL) of the first Fab fragment connected to a VLCH1 chain of the second Fab fragment by a peptide linker (VLCL-linker-VHCH1), and the heavy chain of the third fragment connected to the N-terminus of the VHCL chain of the second Fab fragment, and the C-terminus of said VHCL chain connected to the N-terminus of the heavy chain of the first Fab fragment via a peptide linker (VHCH1-linker-VHCL-linker-VHCH1). In another embodiment said third Fab fragment is connected to the second Fab fragment by two linkers, and the bispecific antibody comprises two chains: a light chain (VLCL) of the first Fab fragment connected to a VLCH1 chain of the second Fab fragment by a peptide linker, which is in turn connected to the light chain of the third Fab fragment (VLCL-linker-VHCH1-linker-VLCL), and the heavy chain of the third fragment connected to the N-terminus of the VHCL chain of the second Fab fragment, and the C-terminus of said VHCL chain connected to the N-terminus of the heavy chain of the first Fab fragment via a peptide linker (VHCH1-linker-VHCL-linker-VHCH1).

B. Exemplary Bispecific Antibodies that Bind to a T-cell Activating Antigen and a Tumor Antigen (TA)

The present invention relates to bispecific antibodies combining a T-cell activating antigen binding site with a second antigen binding site that targets a Tumor Antigen (TA). The antibodies of the invention specifically bind to a Tumor Antigen on the surface of a tumor cell and at the same time bind to an antigen on the surface of cytotoxic T lymphocytes. Preferably said antigen is a CD3 T-Cell Co-Receptor (CD3) antigen. The bispecific antibody is capable to elicit an immune response specifically at the site of the tumor, subsequently resulting in apoptosis of the target cell. It is understood that the sequences exemplified in this section can be arranged in any antibody format exemplified in the embodiments in section A above.

In a particular embodiment according to the invention, the T cell activating bispecific antibody is capable of simultaneous binding to a tumor cell antigen, and an activating T cell antigen. In one embodiment, the T cell activating bispecific antibody is capable of crosslinking a cell and a tumor cell by simultaneous binding to a tumor cell antigen and an activating T cell antigen. In an even more particular embodiment, such simultaneous binding results in lysis of the tumor cell. In one embodiment, such simultaneous binding results in activation of the T cell. In other embodiments, such simultaneous binding results in a cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from the group of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. In one embodiment, binding of the T cell activating bispecific antibody to the activating T cell antigen without simultaneous binding to the target cell antigen does not result in cell activation.

In one embodiment, the T cell activating bispecific antibody is capable of re-directing cytotoxic activity of a T cell to a target cell. In a particular embodiment, said re-direction is independent of MHC-mediated peptide antigen presentation by the target cell and/or specificity of the T cell. Particularly, a T cell according to any of the embodiments of the invention is a cytotoxic T cell. In some embodiments the T cell is a CD4⁺ or a CD8⁺ T cell, particularly a CD8⁺ T cell.

In one embodiment bispecific antibodies are provided that specifically bind a T-cell activating antigen and a Tumor Antigen (TA), comprising a first Fab fragment and a second Fab fragment, wherein either the variable regions or the constant regions of the second Fab heavy and light chain are exchanged and the two Fab fragments are connected to each other by two peptide linkers and wherein the bispecific antibody does not comprise a Fc domain, as detailed in section A above.

In one aspect, a bispecific antibody that specifically binds a T-cell activating antigen and a Tumor Antigen (TA) is provided, comprising at least two Fab fragments connected by two peptide linkers, wherein the first Fab fragment comprises at least one antigen binding site specific for a Tumor Antigen (TA); and the second Fab fragment comprises at least one antigen binding site specific for a T-cell activating antigen, wherein either the variable regions or the constant regions of the second Fab heavy and light chain are exchanged; and wherein the bispecific antibody is devoid of a Fc domain.

In a particular embodiment the T cell activating antigen is the CD3 T-Cell Co-Receptor (CD3) antigen, particularly human or cynomolgus CD3, most particularly human CD3. In some embodiments, the T cell activating antigen is the epsilon subunit of CD3. In other embodiments, the T cell activating antigen is the alpha or beta subunit of CD3.

In one aspect, a bispecific antibody that specifically binds CD3 T-Cell Co-Receptor (CD3) antigen and a Tumor Antigen (TA) is provided, comprising at least two Fab fragments which are connected by two peptide linkers, wherein the first Fab fragment comprises at least one antigen binding site specific for a Tumor Antigen (TA); and the second Fab fragment comprises at least one antigen binding site specific for a CD3 T-Cell Co-Receptor (CD3) wherein either the variable regions or the constant regions of the second Fab heavy and light chain are exchanged; and wherein the bispecific antibody is devoid of a Fc domain.

The bispecific antibodies according to the invention are at least bivalent and can be trivalent or multivalent e.g. tetravalent or hexavalent. In one embodiment said bispecific antibodies are bivalent (1+1 format) with one binding site each targeting a Tumor Antigen (TA) and a T-cell activating antigen, respectively. In another embodiment said bispecific antibodies are trivalent (2+1 format) with two binding sites each targeting a Tumor Antigen (TA) and one binding site targeting a T-cell activating antigen, as detailed in the following section.

In one embodiment said antibody additionally comprises a third Fab fragment, as detailed in section A above. In one embodiment said third Fab fragment comprises at least one antigen binding site specific for a Tumor Antigen. In one embodiment the antigen binding site of said third Fab fragment is specific for the same Tumor Antigen as the antigen binding site of the first Fab fragment.

In one embodiment, the antigen binding site of said third Fab fragment is specific for the same Tumor Antigen as the antigen binding site of the first Fab fragment, and the bispecific antibody of the invention comprises three Fab fragments connected by peptide linkers in the following order (either from N-terminal to C-terminal direction or from C-terminal to N-terminal direction): Fab_((TA))-(1 or 2 linkers)-Fab_((TA))-(2 linkers)-xFab_((T-cell activating antigen)), wherein Fab_((TA)) denotes a Fab fragment with antigen binding site specific for a Tumor Antigen and xFab_((T-cell activating antigen)) denotes a Fab fragment with antigen binding site specific for a T-cell activating antigen, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged.

In one embodiment, the antigen binding site of said third Fab fragment is specific for the same Tumor Antigen as the antigen binding site of the first Fab fragment, and the bispecific antibody of the invention comprises three Fab fragments connected by peptide linkers in the following order (either from N-terminal to C-terminal direction or from C-terminal to N-terminal direction): Fab_((TA))-(2 linkers)-xFab_((T-cell activating antigen))-(1 or 2 linkers)-Fab_((TA)), wherein Fab_((TA)) denotes a Fab fragment with antigen binding site specific for a Tumor Antigen and xFab_((T-cell activating antigen)) denotes a Fab fragment with antigen binding site specific for a T-cell activating antigen, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged.

In one embodiment the bispecific antibody comprises an antigen binding moiety that can compete with monoclonal antibody V9 for binding to an epitope of CD3. See for example Rodigues et al., Int J Cancer Suppl 7 (1992), 45-50; U.S. Pat. No. 6,054,297, incorporated herein by reference in its entirety.

In one embodiment the bispecific antibody comprises an antigen binding moiety that can compete with monoclonal antibody FN18 for binding to an epitope of CD3. See Nooij et al., Eur J Immunol 19 (1986), 981-984, incorporated herein by reference in its entirety.

In one embodiment the bispecific antibody comprises an antigen binding moiety that can compete with monoclonal antibody CH2527 (Sequence ID 157 and 158 and humanized variants) or an affinity matured variant thereof for binding to an epitope of CD3.

In one embodiment the bispecific antibody comprises a second Fab fragment specifically binding to CD3, wherein the heavy chain variable region comprises a CDR1 of SEQ ID. NO. 10 or SEQ ID. NO. 32, a CDR2 of SEQ ID. NO. 11 or SEQ ID. NO. 33, and a CDR3 of SEQ ID. NO. 12 or SEQ ID. NO. 34; and wherein the light chain variable region comprises a CDR1 of SEQ ID. NO. 7 or SEQ ID. NO. 29, a CDR2 of SEQ ID. NO. 8 or SEQ ID. NO. 30, and a CDR3 of SEQ ID. NO. 9 or SEQ ID. NO.31.

In one embodiment the bispecific antibody comprises a second Fab fragment specifically binding to CD3, wherein the heavy chain variable region comprises a CDR1 of SEQ ID. NO. 10, a CDR2 of SEQ ID. NO. 11, and a CDR3 of SEQ ID. NO. 12; and wherein the light chain variable region comprises a CDR1 of SEQ ID. NO. 7, a CDR2 of SEQ ID. NO. 8 and a CDR3 of SEQ ID. NO. 9.

In one embodiment the bispecific antibody comprises a second Fab fragment specifically binding to CD3, wherein the heavy chain variable region comprises a CDR1 of SEQ ID. NO. 32, a CDR2 SEQ ID. NO. 33, and a CDR3 of SEQ ID. NO. 34; and wherein the light chain variable region comprises a CDR1 of SEQ ID. NO. 29, a CDR2 of SEQ ID. NO. 30, and a CDR3 of SEQ ID. NO.31.

In one embodiment the bispecific antibody comprises a second Fab fragment specifically binding to CD3, wherein the heavy chain variable region comprises a CDR1 of SEQ ID. NO. 170, a CDR2 of SEQ ID. NO. 171, and a CDR3 of SEQ ID. NO. 172; and wherein the light chain variable region comprises a CDR1 of SEQ ID. NO. 174, a CDR2 of SEQ ID. NO. 175 and a CDR3 of SEQ ID. NO. 176.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein the heavy chain variable region sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID. NO. 20 or SEQ ID. NO. 36; wherein the light chain variable region sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID. NO. 19. or SEQ ID. NO 35, or variants thereof that retain functionality. In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID. NO. 20; and a light chain variable region comprising an amino acid sequence of SEQ ID. NO. 19 or variants thereof that retain functionality.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID. NO. 36; and a light chain variable region comprising an amino acid sequence of SEQ ID. NO. 35 or variants thereof that retain functionality.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID. NO. 158; and a light chain variable region comprising an amino acid sequence of SEQ ID. NO. 157 or variants thereof that retain functionality. In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein the heavy chain variable region sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID. NO. 158; wherein the light chain variable region sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID. NO. 157, or variants thereof that retain functionality. In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein the heavy chain variable region sequence is an affinity matured variant of SEQ ID. NO. 158 and wherein the light chain variable region sequence is an affinity matured variant of SEQ ID. NO. 157. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 158 and/or SEQ ID. NO. 157 are exchanged.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID. NO. 169; and a light chain variable region comprising an amino acid sequence of SEQ ID. NO. 173.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of second Fab fragment specifically binding to CD3, wherein said heavy chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 22 or SEQ ID. NO. 38 or variants thereof that retain functionality. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein said heavy chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 22 or SEQ ID. NO 38, and a light chain and a heavy chain of first Fab fragment specific for a Tumor Antigen (TA) comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of 15 second Fab fragment specifically binding to CD3, wherein said heavy chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 22. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein said heavy chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 22, and a light chain and a heavy chain of first Fab fragment specific for a Tumor Antigen (TA) comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein said light chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID. NO. 37. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein said light chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID. NO. 37, and a light chain and a heavy chain of a first Fab fragment specific for a Tumor Antigen (TA) comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein said light chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 21. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, wherein said light chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 21, and a light chain and a heavy chain of a first Fab fragment specific for a Tumor Antigen (TA) comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In yet another specific embodiment, a bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, said heavy chain comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 22 or SEQ ID. NO. 38; and said light chain comprising a light chain constant region comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID. NO. 37.

In yet another specific embodiment, a bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, said heavy chain comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 22; and said light chain comprising a light chain constant region comprising the amino acid sequence of SEQ ID NO: 21.

In yet another specific embodiment, a bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, said heavy chain comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 22; and said light chain comprising a light chain constant region comprising the amino acid sequence of SEQ ID NO: 21, and a light chain and a heavy chain of a first Fab fragment specific for a Tumor Antigen (TA) comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 22, and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 21.

In one embodiment the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 22, and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 21, and a light chain and a heavy chain of a first Fab fragment specific for a Tumor Antigen (TA) comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 35 and a variable heavy chain of SEQ ID NO: 36, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 38, and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 37.

In one embodiment the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 35 and a variable heavy chain of SEQ ID NO: 36, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 38, and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 37., and a light chain and a heavy chain of a first Fab fragment specific for a Tumor Antigen (TA) comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 173 and a variable heavy chain of SEQ ID NO: 169, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 38, and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 37.

In one embodiment the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 173 and a variable heavy chain of SEQ ID NO: 169, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 38, and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 37., and a light chain and a heavy chain of a first Fab fragment specific for a Tumor Antigen (TA) comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 157 and a variable heavy chain of SEQ ID NO: 158, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 22, and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 21.

In one embodiment the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 157 or an affinity matured variant thereof and a variable heavy chain of SEQ ID NO: 158 or an affinity matured variant thereof, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 22, and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 21. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 158 and/or SEQ ID. NO. 157 are exchanged.

In one embodiment the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 157 and a variable heavy chain of SEQ ID NO: 158, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 22, and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 21, and a light chain and a heavy chain of a first Fab fragment specific for a Tumor Antigen (TA) comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 157 or an affinity matured variant thereof and a variable heavy chain of SEQ ID NO: 158 or an affinity matured variant thereof, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 22, and a light chain constant region comprising the amino acid sequence of SEQ ID NO: 21 and a heavy chain of a first Fab fragment specific for a Tumor Antigen (TA) comprising one or more amino acid sequences as defined in any of the embodiments described herein. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 158 and/or SEQ ID. NO. 157 are exchanged.

In one embodiment the Tumor Antigen is selected from the group of Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), Carcinoembryonic Antigen (CEA), Fibroblast Activation Protein (FAP) and CD33. In one preferred embodiment the Tumor Antigen is MCSP.

In one embodiment the T cell activating bispecific antibody comprises at least one antigen binding site that is specific for Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP). In another embodiment the T cell activating bispecific antibody comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody M4-3 ML2 (Sequence ID 161 and 162) or an affinity matured variant thereof for binding to an epitope of MCSP.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to MCSP, wherein the variable heavy chain comprises a CDR1 of SEQ ID. NO. 4, a CDR2 of SEQ ID. NO. 5, a CDR3 of SEQ ID. NO. 6; and the variable light chain comprises a CDR1 of SEQ ID. NO. 1, a CDR2 of SEQ ID. NO.2, and a CDR3 of SEQ ID. NO. 3.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to MCSP, wherein the variable heavy chain comprises a CDR1 of SEQ ID. NO. 180, a CDR2 of SEQ ID. NO. 181, a CDR3 of SEQ ID. NO. 6; and the variable light chain comprises a CDR1 of SEQ ID. NO. 183, a CDR2 of SEQ ID. NO.2, and a CDR3 of SEQ ID. NO. 184.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to MCSP, wherein the heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID. NO. 14; and a light chain variable region is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID. NO. 13.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to MCSP, wherein the heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID. NO. 161; and a light chain variable region is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID. NO. 162.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to MCSP, wherein the heavy chain variable region sequence is an affinity matured variant of SEQ ID. NO. 161 and wherein the light chain variable region sequence is an affinity matured variant of SEQ ID. NO. 162. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 161 and/or SEQ ID. NO. 162 are exchanged.

In one embodiment said affinity matured variant comprises a variable heavy chain comprising a CDR1 of SEQ ID. NO. 180, a CDR2 of SEQ ID. NO. 181, a CDR3 of SEQ ID. NO. 6; and a variable light chain comprising a CDR1 of SEQ ID. NO. 183, a CDR2 of SEQ ID. NO.2, and a CDR3 of SEQ ID. NO. 184. In another embodiment said affinity matured variant comprises a heavy chain variable region sequence of SEQ ID. NO. 179 and wherein the light chain variable region sequence is an affinity matured variant of SEQ ID. NO. 182.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to MCSP, wherein said heavy chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 16. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to MCSP, wherein said heavy chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 16, and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to MCSP, wherein said light chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 15. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a second antibody specifically binding to MCSP, wherein said light chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 15, and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to MCSP, wherein the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 16; and a light chain constant region comprising an amino acid sequence of SEQ ID NO: 15.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to MCSP, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 14; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 13, and wherein the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 16; and a light chain constant region comprising an amino acid sequence of SEQ ID NO: 15.

In a further embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20; and a light chain and a heavy chain of a first Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 13 and a variable heavy chain of SEQ ID NO: 14.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to MCSP, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 161; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 162, and wherein the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 16; and a light chain constant region comprising an amino acid sequence of SEQ ID NO: 15.

In a further embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20; and a light chain and a heavy chain of a first Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 161 and a variable heavy chain of SEQ ID NO: 162.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to MCSP, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 161 or an affinity matured variant thereof; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 162 or an affinity matured variant thereof, and wherein the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 16; and a light chain constant region comprising an amino acid sequence of SEQ ID NO: 15. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 161 and/or SEQ ID. NO. 162 are exchanged.

In a further embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20; and a light chain and a heavy chain of a first Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 161 or an affinity matured variant thereof and a variable heavy chain of SEQ ID NO: 162 or an affinity matured variant thereof. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 161 and/or SEQ ID. NO. 162 are exchanged.

In a further embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 158 and a variable heavy chain of SEQ ID NO: 157; and a light chain and a heavy chain of a first Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 161 and a variable heavy chain of SEQ ID NO: 162.

In a further embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 158 or an affinity matured variant thereof and a variable heavy chain of SEQ ID NO: 157 or an affinity matured variant thereof; and a light chain and a heavy chain of a first Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 161 or an affinity matured variant thereof and a variable heavy chain of SEQ ID NO: 162 or an affinity matured variant thereof. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of one or more of SEQ ID. NO. 157, SEQ ID. NO. 158, SEQ ID. NO. 161 and/or SEQ ID. NO. 162 are exchanged.

In one embodiment the bispecific antibody comprises a third Fab fragment, comprising a light chain and a heavy chain specifically binding to MCSP, wherein the variable heavy chain comprises a CDR1 of SEQ ID. NO. 4, a CDR2 of SEQ ID. NO. 5, a CDR3 of SEQ ID. NO. 6; and the variable light chain comprises a CDR1 of SEQ ID. NO. 1, a CDR2 of SEQ ID. NO.2, and a CDR3 of SEQ ID. NO. 3.

In one embodiment the bispecific antibody comprises a third Fab fragment, comprising a a light chain and a heavy chain specifically binding to MCSP, wherein the heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID. NO. 14; and a light chain variable region is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID. NO. 13.

In one embodiment the bispecific antibody comprises a third Fab fragment, comprising a a light chain and a heavy chain specifically binding to MCSP, wherein the heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID. NO. 161; and a light chain variable region is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID. NO. 162.

In one embodiment the bispecific antibody comprises a third Fab fragment, comprising a a light chain and a heavy chain specifically binding to MCSP, wherein the heavy chain variable region sequence is an affinity matured variant of SEQ ID. NO. 161 and wherein the light chain variable region sequence is an affinity matured variant of SEQ ID. NO. 162. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 161 and/or SEQ ID. NO. 162 are exchanged.

In one embodiment the bispecific antibody comprises a third Fab fragment, comprising a light chain and a heavy chain specifically binding to MCSP, wherein said heavy chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 16. In one embodiment the bispecific antibody comprises a third Fab fragment, comprising a light chain and a heavy chain specifically binding to MCSP, wherein said heavy chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 16, and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more amino acid sequences as defined in any of the embodiments described herein, and a a light chain and a heavy chain of a first Fab fragment specific for MCSP comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment the bispecific antibody comprises a third Fab fragment, comprising a light chain and a heavy chain specifically binding to MCSP, wherein said light chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 15. In one embodiment, the bispecific antibody comprises a light chain and a heavy chain of a second antibody specifically binding to MCSP, wherein said light chain comprises a constant region comprising the amino acid sequence of SEQ ID NO: 15, and a light chain and a heavy chain of a second Fab fragment specific for CD3, and a light chain and a heavy chain of a first Fab fragment specific for MCSP comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment the bispecific antibody comprises a third Fab fragment, comprising a light chain and a heavy chain specifically binding to MCSP, wherein the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 16; and a light chain constant region comprising an amino acid sequence of SEQ ID NO: 15.

In one embodiment the bispecific antibody comprises a third Fab fragment, comprising a light chain and a heavy chain specifically binding to MCSP, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 14; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 13, and wherein the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 16; and a light chain constant region comprising an amino acid sequence of SEQ ID NO: 15.

In one embodiment the bispecific antibody comprises a third Fab fragment, comprising a light chain and a heavy chain specifically binding to MCSP, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 161; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 162, and wherein the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 16; and a light chain constant region comprising an amino acid sequence of SEQ ID NO: 15.

In one embodiment the bispecific antibody comprises a third Fab fragment, comprising a light chain and a heavy chain specifically binding to MCSP, wherein the heavy chain variable region sequence is an affinity matured variant of SEQ ID. NO. 161 and wherein the light chain variable region sequence is an affinity matured variant of SEQ ID. NO. 162, and wherein the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 16; and a light chain constant region comprising an amino acid sequence of SEQ ID NO: 15. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 161 and/or SEQ ID. NO. 162 are exchanged.

In a further embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20; and a light chain and a heavy chain of a first Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 13 and a variable heavy chain of SEQ ID NO: 14, and a light chain and a heavy chain of a third Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 13 and a variable heavy chain of SEQ ID NO: 14.

In a further embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20; and a light chain and a heavy chain of a first Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 162 and a variable heavy chain of SEQ ID NO: 161, and a light chain and a heavy chain of a third Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 162 and a variable heavy chain of SEQ ID NO: 161.

In a further embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 19 and a variable heavy chain of SEQ ID NO: 20; and a light chain and a heavy chain of a first Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 162 or an affinity matured variant thereof and a variable heavy chain of SEQ ID NO: 161 or an affinity matured variant thereof, and a light chain and a heavy chain of a third Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 162 or an affinity matured variant thereof and a variable heavy chain of SEQ ID NO: 161 or an affinity matured variant thereof. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 161 and/or SEQ ID. NO. 162 are exchanged.

In a further embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 157 and a variable heavy chain of SEQ ID NO: 158; and a light chain and a heavy chain of a first Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 162 and a variable heavy chain of SEQ ID NO: 161, and a light chain and a heavy chain of a third Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 162 and a variable heavy chain of SEQ ID NO: 161.

In a further embodiment, the bispecific antibody of the invention comprises a light chain and a heavy chain of a second Fab fragment specifically binding to CD3, comprising a variable light chain of SEQ ID NO: 157 and a variable heavy chain of SEQ ID NO: 158; and a light chain and a heavy chain of a first Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 162 or an affinity matured variant thereof and a variable heavy chain of SEQ ID NO: 161 or an affinity matured variant thereof, and a light chain and a heavy chain of a third Fab fragment specific for MCSP, comprising a variable light chain of SEQ ID NO: 162 or an affinity matured variant thereofand a variable heavy chain of SEQ ID NO: 161 or an affinity matured variant thereof. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 161 and/or SEQ ID. NO. 162 are exchanged.

In yet another embodiment said bispecific antibody comprises one or more amino acid sequences selected from the group of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO. 41 and SEQ ID NO. 43.

In one embodiment said bispecific antibody comprises SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27.

In one preferred embodiment said bispecific antibody comprises SEQ ID NO: 163 and, SEQ ID NO: 164.

In one embodiment the T cell activating bispecific antibody comprises at least one antigen binding site that is specific for Epidermal Growth Factor Receptor (EGFR). In another embodiment the T cell activating bispecific antibody comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody GA201 for binding to an epitope of EGFR. See PCT publication WO 2006/082515, incorporated herein by reference in its entirety. In one embodiment, the antigen binding site that is specific for EGFR comprises the heavy chain CDR1 of SEQ ID NO: 68, the heavy chain CDR2 of SEQ ID NO: 69, the heavy chain CDR3 of SEQ ID NO: 70, the light chain CDR1 of SEQ ID NO: 71, the light chain CDR2 of SEQ ID NO: 72, and the light chain CDR3 of SEQ ID NO: 73. In a further embodiment, the antigen binding site that is specific for EGFR comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 74 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 75, or variants thereof that retain functionality.

In a further embodiment, the bispecific antibody comprises a first Fab fragment comprising an antigen binding site that is specific for EGFR comprising a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 74 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 75, or variants thereof that retain functionality, and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In a further embodiment, the bispecific antibody comprises a first and a third Fab fragment comprising an antigen binding site that is specific for EGFR comprising a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 74 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 75, or variants thereof that retain functionality, and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment the T cell activating bispecific antibody comprises at least one antigen binding site that is specific for Fibroblast Activation Protein (FAP). In another embodiment the T cell activating bispecific antibody comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody 3F2 for binding to an epitope of FAP. See European patent application no. EP10172842.6, incorporated herein by reference in its entirety. In one embodiment, the antigen binding site that is specific for FAP comprises the heavy chain CDR1 of SEQ ID NO: 76, the heavy chain CDR2 of SEQ ID NO: 77, the heavy chain CDR3 of SEQ ID NO: 78, the light chain CDR1 of SEQ ID NO: 79, the light chain CDR2 of SEQ ID NO: 80, and the light chain CDR3 of SEQ ID NO: 81. In a further embodiment, the antigen binding site that is specific for FAP comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 82 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 83, or variants thereof that retain functionality.

In a further embodiment, the bispecific antibody comprises a first Fab fragment comprising an antigen binding site that is specific for FAP comprising a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 82 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 83, or variants thereof that retain functionality, and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In a further embodiment, the bispecific antibody comprises a first and a third Fab fragment comprising an antigen binding site that is specific for FAP comprising a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 82 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 83, or variants thereof that retain functionality, and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment the T cell activating bispecific antibody comprises at least one antigen binding site that is specific for Carcinoembryonic Antigen (CEA). In another embodiment the T cell activating bispecific antibody comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody CH1A1A for binding to an epitope of CEA. In one embodiment the T cell activating bispecific antibody comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody CH1A1A clone 98/99 (CH1A1_((98/99))) for binding to an epitope of CEA. See PCT patent application number PCT/EP2010/062527, incorporated herein by reference in its entirety. In one embodiment, the antigen binding site that is specific for CEA comprises the heavy chain CDR1 of SEQ ID NO: 84, the heavy chain CDR2 of SEQ ID NO: 85, the heavy chain CDR3 of SEQ ID NO: 86, the light chain CDR1 of SEQ ID NO: 87, the light chain CDR2 of SEQ ID NO: 88, and the light chain CDR3 of SEQ ID NO: 89. In a further embodiment, the antigen binding site that is specific for CEA comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 90 or SEQ ID NO: 159 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 91 or SEQ ID NO: 160, or variants thereof that retain functionality.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to CEA, wherein the heavy chain variable region comprises an affinity matured variant of SEQ ID NO: 159 or thereof; and a light chain variable region comprising an affinity matured variant of SEQ ID NO: 160. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 159 and/or SEQ ID. NO. 160 are exchanged.

In a further embodiment, the bispecific antibody comprises a first Fab fragment comprising an antigen binding site that is specific for CEA comprising a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 90 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 91, or variants thereof that retain functionality, and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In a further embodiment, the bispecific antibody comprises a first Fab fragment comprising an antigen binding site that is specific for CEA comprising a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 159 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 160, or variants thereof that retain functionality, and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In one embodiment the bispecific antibody comprises a light chain and a heavy chain of a first Fab fragment specifically binding to CEA, wherein the heavy chain variable region comprises an affinity matured variant of SEQ ID NO: 159; and a light chain variable region comprising an affinity matured variant of SEQ ID NO: 160 and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more amino acid sequences as defined in any of the embodiments described herein. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 159 and/or SEQ ID. NO. 160 are exchanged.

In a further embodiment, the bispecific antibody comprises a first and a third Fab fragment comprising an antigen binding site that is specific for CEA comprising a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 90 or SEQ ID NO: 159 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 91 or SEQ ID NO: 160, or variants thereof that retain functionality, and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more amino acid sequences as defined in any of the embodiments described herein. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 159 and/or SEQ ID. NO. 160 are exchanged.

In a further embodiment, the bispecific antibody comprises a first and a third Fab fragment comprising an antigen binding site that is specific for CEA wherein the heavy chain variable region comprises an affinity matured variant of SEQ ID NO: 159; and the light chain variable region comprising an affinity matured variant of SEQ ID NO: 160. Affinity matured variants in this embodiment means that independently 1, 2, 3 or 4 amino acids of SEQ ID. NO. 159 and/or SEQ ID. NO. 160 are exchanged.

In one embodiment the T cell activating bispecific antibody comprises at least one antigen binding site that is specific for CD33. In one embodiment, the antigen binding site that is specific for CD33 comprises the heavy chain CDR1 of SEQ ID NO: 92, the heavy chain CDR2 of SEQ ID NO: 93, the heavy chain CDR3 of SEQ ID NO: 94, the light chain CDR1 of SEQ ID NO: 95, the light chain CDR2 of SEQ ID NO: 96, and the light chain CDR3 of SEQ ID NO: 97. In a further embodiment, the antigen binding site that is specific for CD33 comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 98 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 99, or variants thereof that retain functionality.

In a further embodiment, the bispecific antibody comprises a first Fab fragment comprising an antigen binding site that is specific for CD33 comprising a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 98 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 99, or variants thereof that retain functionality, and a light chain and a heavy chain of a second Fab fragment specific for CD3 comprising one or more amino acid sequences as defined in any of the embodiments described herein.

In a specific embodiment the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102.

In one embodiment the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 151, SEQ ID NO. 152 and SEQ ID NO. 153.

In yet another embodiment said bispecific antibody comprises one or more amino acid sequences selected from the group of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 151, SEQ ID NO. 152 and SEQ ID NO. 153.

In one embodiment of the invention the bispecific antibody is a humanized antibody, as detailed below.

In another embodiment of the invention the bispecific antibody is a human antibody, as detailed below.

In a second object the present invention relates to a pharmaceutical composition comprising a bispecific antibody of the present invention.

In a third object the present invention relates to a bispecific antibody of the present invention for the treatment of cancer. In another embodiment, use of the bispecific antibody as a medicament is provided. Preferably said use is for the treatment of cancer.

In further objects the present invention relates to a nucleic acid sequence comprising a sequence encoding a heavy chain of a bispecific antibody of the present invention, a nucleic acid sequence comprising a sequence encoding a light chain of a bispecific antibody of the present invention, an expression vector comprising a nucleic acid sequence of the present invention and to a prokaryotic or eukaryotic host cell comprising a vector of the present invention. In addition a method of producing an antibody comprising culturing the host cell so that the antibody is produced is provided.

In a further embodiment an immunoconjugate comprising the bispecific antibody of the invention and a cytotoxic agent is provided.

In further objects the present invention relates to a nucleic acid sequence comprising a sequence encoding a heavy chain of a bispecific antibody of the present invention, a nucleic acid sequence comprising a sequence encoding a light chain of a bispecific antibody of the present invention, an expression vector comprising a nucleic acid sequence of the present invention and to a prokaryotic or eukaryotic host cell comprising a vector of the present invention. In addition a method of producing an antibody comprising culturing the host cell so that the antibody is produced is provided.

In a specific embodiment the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

In a specific embodiment the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112.

In a specific embodiment the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 120.

In a specific embodiment the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, and SEQ ID NO: 128.

In a specific embodiment the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, and SEQ ID NO: 136.

In a specific embodiment the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 156.

In a specific embodiment the T cell activating bispecific antibody comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 165 and SEQ ID NO: 166.

In a further aspect, a bispecific antibody according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 1-5 below:

1. Antibody Affinity

The affinity of the bispecific antibody provided herein for a target antigen can be determined in accordance with the methods set forth in the Examples by surface plasmon resonance (SPR), using standard instrumentation such as a BIACORE® instrument (GE Healthcare), and receptors or target proteins such as may be obtained by recombinant expression. Alternatively, binding of bispecific antibodies for different receptors or target antigens may be evaluated using cell lines expressing the particular receptor or target antigen, for example by flow cytometry (FACS).

In certain embodiments, a bispecific antibody provided herein has a dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M).

According to one embodiment, KD is measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at ˜10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5 μl/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately 25 μl/min. Association rates (ka or k_(on)) and dissociation rates (kd or k_(off)) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is calculated as the ratio k_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Chimeric and Humanized Antibodies

In certain embodiments, a bispecific antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall' Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

3. Human Antibodies

In certain embodiments, a bispecific antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

4. Library-Derived Antibodies

Bispecific antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N. J., 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N. J., 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

5. Antibody Variants

In certain embodiments, amino acid sequence variants of the bispecific antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the bispecific antibody. Amino acid sequence variants of a bispecific antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the bispecific antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 2 under the heading of “conservative substitutions.” More substantial changes are provided in Table 2 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding or decreased immunogenicity.

TABLE 2 Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots” or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

b) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteine engineered bispecific antibodies, e.g., “thioMAbs,” in which one or more residues of a bispecific antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the bispecific antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain and A118 (EU numbering) of the heavy chain. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521,541.

c) Antibody Derivatives

In certain embodiments, a bispecific antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the bispecific antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of a bispecific antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.

C. Recombinant Methods and Compositions

Bispecific antibodies of the invention may be obtained, for example, by solid-state peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production. For recombinant production one or more polynucleotide encoding the bispecific anitbody (fragment), e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotide may be readily isolated and sequenced using conventional procedures. In one embodiment a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention is provided. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of a bispecific antibody (fragment) along with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y (1989). The expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which the polynucleotide encoding the bispecific antibody (fragment) (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation control elements. As used herein, a “coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5′ and 3′ untranslated regions, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g. a vector of the present invention may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage. In addition, a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the bispecific antibody (fragment) of the invention, or variant or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable association is when a coding region for a gene product, e.g. a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are “operably associated” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions, which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit â-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as inducible promoters (e.g. promoter inducible tetracyclins). Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence). The expression cassette may also include other features such as an origin of replication, and/or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno-associated viral (AAV) inverted terminal repeats (ITRs).

Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. For example, if secretion of the bispecific antigen binding molecule is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding a bispecific antibody of the invention or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or “mature” form of the polypeptide. In certain embodiments, the native signal peptide, e.g. an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the bispecific antibody may be included within or at the ends of the bispecific antibody (fragment) encoding polynucleotide.

In a further embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In certain embodiments a host cell comprising one or more vectors of the invention is provided. The polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively. In one such embodiment a host cell comprises (e.g. has been transformed or transfected with) a vector comprising a polynucleotide that encodes (part of) a bispecific antibody of the invention. As used herein, the term “host cell” refers to any kind of cellular system which can be engineered to generate the bispecific antibodies of the invention or fragments thereof. Host cells suitable for replicating and for supporting expression of bispecific antibodies are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the bispecific antibody for clinical applications. Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, or the like. For example, polypeptides may be produced in bacteria in particular when glycosylation is not needed. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of a polypeptide with a partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See e.g. U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr⁻ CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as Y0, NS0, P3X63 and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). Standard technologies are known in the art to express foreign genes in these systems. Cells expressing a polypeptide comprising either the heavy or the light chain of an antigen binding domain such as an antibody, may be engineered so as to also express the other of the antibody chains such that the expressed product is an antibody that has both a heavy and a light chain. In one embodiment, a method of producing a bispecific antibody according to the invention is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding the bispecific antibody, as provided herein, under conditions suitable for expression of the bispecific antigen binding molecule, and recovering the bispecific antibody from the host cell (or host cell culture medium).

The components of the bispecific antibody are genetically fused to each other. bispecific antibody can be designed such that its components are fused directly to each other or indirectly through a linker sequence. The composition and length of the linker may be determined in accordance with methods well known in the art and may be tested for efficacy. Examples of linker sequences between different components of bispecific antibodies are found in the sequences provided herein. Additional sequences may also be included to incorporate a cleavage site to separate the individual components of the fusion if desired, for example an endopeptidase recognition sequence.

In certain embodiments the one or more antigen binding moieties of the bispecific antibodies comprise at least an antibody variable region capable of binding an antigenic determinant. Variable regions can form part of and be derived from naturally or non-naturally occurring antibodies and fragments thereof. Methods to produce polyclonal antibodies and monoclonal antibodies are well known in the art (see e.g. Harlow and Lane, “Antibodies, a laboratory manual”, Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase-peptide synthesis, can be produced recombinantly (e.g. as described in U.S. Pat. No. 4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy chains and variable light chains (see e.g. U.S. Pat. No. 5,969,108 to McCafferty).

Any animal species of antibody, antibody fragment, antigen binding domain or variable region can be used in the bispecific antibodies of the invention. Non-limiting antibodies, antibody fragments, antigen binding domains or variable regions useful in the present invention can be of murine, primate, or human origin. If the antibody is intended for human use, a chimeric form of antibody may be used wherein the constant regions of the antibody are from a human. A humanized or fully human form of the antibody can also be prepared in accordance with methods well known in the art (see e. g. U.S. Pat. No. 5,565,332 to Winter). Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions), (b) grafting only the non-human specificity-determining regions (SDRs or a-CDRs; the residues critical for the antibody-antigen interaction) onto human framework and constant regions, or (c) transplanting the entire non-human variable domains, but “cloaking” them with a human-like section by replacement of surface residues. Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing “resurfacing”); Dall' Acqua et al., Methods 36, 43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000) (describing the “guided selection” approach to FR shuffling). Human antibodies and human variable regions can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regions can form part of and be derived from human monoclonal antibodies made by the hybridoma method (see e.g. Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions may also be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge (see e.g. Lonberg, Nat Biotech 23, 1117-1125 (2005). Human antibodies and human variable regions may also be generated by isolating Fv clone variable region sequences selected from human-derived phage display libraries (see e.g., Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien et al., ed., Human Press, Totowa, N. J., 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.

In certain embodiments, the bispecific antibodies of the present invention are engineered to have enhanced binding affinity according to, for example, the methods disclosed in U.S. Pat. Appl. Publ. No. 2004/0132066, the entire contents of which are hereby incorporated by reference. The ability of the bispecific antibody of the invention to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance technique (analyzed on a BIACORE® T100 system) (Liljeblad, et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays may be used to identify an antibody, antibody fragment, antigen binding domain or variable domain that competes with a reference antibody for binding to a particular antigen, e.g. an antibody that competes with the V9 antibody for binding to CD3. In certain embodiments, such a competing antibody binds to the same epitope (e.g. a linear or a conformational epitope) that is bound by the reference antibody. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.). In an exemplary competition assay, immobilized antigen (e.g. CD3) is incubated in a solution comprising a first labeled antibody that binds to the antigen (e.g. V9 antibody) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the antigen. The second antibody may be present in a hybridoma supernatant. As a control, immobilized antigen is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to the antigen, excess unbound antibody is removed, and the amount of label associated with immobilized antigen is measured. If the amount of label associated with immobilized antigen is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Bispecific antibodies prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the bispecific antibody binds. For example, for affinity chromatography purification of bispecific antibody of the invention, a matrix with protein A or protein G may be used. Sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate a bispecific antibody essentially as described in the Examples. The purity of the bispecific antibodies can be determined by any of a variety of well known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like.

D. Assays

Bispecific antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, a bispecific antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc.

In another aspect, competition assays may be used to identify an antibody that competes with a specific antibody for binding to the first or second antigen. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by a specific antibody. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.).

2. Activity Assays

In one aspect, assays are provided for identifying bispecific antibodies thereof having biological activity. Biological activity may include, e.g., lysis of targeted cells, or induction of apoptosis. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, a bispecific antibody of the invention is tested for such biological activity. Assays for detecting cell lysis (e.g. by measurement of LDH release) or apoptosis (e.g. using the TUNEL assay) are well known in the art.

E. Immunoconjugates

The invention also provides immunoconjugates comprising a bispecific antibody of the invention conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 BI); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC 1065.

In another embodiment, an immunoconjugate comprises a bispecific antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises a bispecific antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of a bispecific antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026. The linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

F. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the bispecific antibodies provided herein is useful for detecting the presence of a first and/or a second antigenin a biological sample. The term “detecting” as used herein encompasses quantitative or qualitative detection. In certain embodiments, a biological sample comprises a cell or tissue.

In one embodiment, a bispecific antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of a first and/or a second antigen in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with a bispecific antibody as described herein under conditions permissive for binding of the bispecific antibody to a first and/or a second antigen, and detecting whether a complex is formed between the bispecific antibody a first and/or a second antigen. Such method may be an in vitro or in vivo method. In one embodiment, a bispecific antibody is used to select subjects eligible for therapy with a bispecific antibody, e.g. where a first and/or a second antigen is a biomarker for selection of patients.

Exemplary disorders that may be diagnosed using an antibody of the invention include cancer.

In certain embodiments, labeled bispecific antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

G. Pharmaceutical Formulations

Pharmaceutical formulations of a bispecific antibody as described herein are prepared by mixing such bispecific antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations including a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

H. Therapeutic Methods and Compositions

Any of the bispecific antibodies provided herein may be used in therapeutic methods.

In one aspect, a bispecific antibody for use as a medicament is provided. In further aspects, a bispecific antibody for use in treating cancer is provided. In certain embodiments, a bispecific antibody for use in a method of treatment is provided. In certain embodiments, the invention provides a bispecific antibody for use in a method of treating an individual having cancer comprising administering to the individual an effective amount of the bispecific antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. An “individual” according to any of the above embodiments is preferably a human.

In a further aspect, the invention provides for the use of a bispecific antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of cancer. In a further embodiment, the medicament is for use in a method of treating cancer comprising administering to an individual having cancer an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. An “individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for treating cancer. In one embodiment, the method comprises administering to an individual having cancer an effective amount of a bispecific antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An “individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides pharmaceutical formulations comprising any of the bispecific antibodies provided herein, e.g., for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the bispecific antibody provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the bispecific antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

The bispecific antibodies of the invention can be used either alone or in combination with other agents in a therapy. For instance, a bispecific antibody of the invention may be co-administered with at least one additional therapeutic agent.

Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. Bispecific antibodies of the invention can also be used in combination with radiation therapy.

A bispecific antibody of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

Bispecific antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The bispecific antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of a bispecific antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the bispecific antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the bispecific antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of bispecific antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the bispecific antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the bispecific antibody). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

It is understood that any of the above formulations or therapeutic methods may be carried out using an immunoconjugate of the invention in place of or in addition to a bispecific antibody of the invention.

I. Articles of Manufacture

In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a bispecific antibody of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a bispecific antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture may include an immunoconjugate of the invention in place of or in addition to a bispecific antibody of the invention.

III. Examples

The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

EXAMPLE 1 Preparation of Fab (MCSP)-CrossFab(CD3)

The resulting variable region of heavy and light chain DNA sequences have were subcloned in frame with either the constant heavy chain or the constant light chain pre-inserted into the respective recipient mammalian expression vector. The antibody expression is driven by an MPSV promoter and carries a synthetic polyA signal sequence at the 3′ end of the CDS. In addition each vector contains an EBV OriP sequence.

The molecule was produced by co-transfecting HEK293-EBNA cells with the mammalian expression vectors using a calcium phosphate-transfection. Exponentially growing HEK293-EBNA cells were transfected by the calcium phosphate method. Alternatively, HEK293-EBNA cells growing in suspension were transfected by polyethylenimine. The cells were transfected with the corresponding expression vectors in a 1:1:1 ratio (“vector CH1-VH-CK-VH”:“vector light chain”:“vector light chain CH1-VL”).

For transfection using calcium phosphate cells were grown as adherent monolayer cultures in T-flasks using DMEM culture medium supplemented with 10% (v/v) FCS, and were transfected when they were between 50 and 80% confluent. For the transfection of a T150 flask, 15 million cells were seeded 24 hours before transfection in 25 ml DMEM culture medium supplemented with FCS (at 10% v/v final), and cells were placed at 37° C. in an incubator with a 5% CO2 atmosphere overnight. For each T150 flask to be transfected, a solution of DNA, CaCl2 and water was prepared by mixing 94 μg total plasmid vector DNA divided in the corresponding ratio, water to a final volume of 469 μl and 469 μl of a 1 M CaCl2 solution. To this solution, 938 μl of a 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4 solution at pH 7.05 were added, mixed immediately for 10 s and left to stand at room temperature for 20 s. The suspension was diluted with 10 ml of DMEM supplemented with 2% (v/v) FCS, and added to the T150 in place of the existing medium. Then additional 13 ml of transfection medium were added. The cells were incubated at 37° C., 5% CO2 for about 17 to 20 hours, then medium was replaced with 25 ml DMEM, 10% FCS. The conditioned culture medium was harvested approx. 7 days post-media exchange by centrifugation for 15 min at 210×g, the solution was sterile filtered (0.22 μm filter) and sodium azide in a final concentration of 0.01% (w/v) was added, and kept at 4° C.

For transfection using polyethylenimine HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 5 min by 210×g, supernatant was replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 μg DNA. After addition of 540 μl PEI solution was vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 (Lonza) was added. After 7 days cultivation supernatant was collected for purification by centrifugation for 15 min at 210×g, the solution was sterile filtered (0.22 μm filter) and sodium azide in a final concentration of 0.01% w/v was added, and kept at 4° C.

The secreted protein was purified from cell culture supernatants by affinity chromatography using Protein A and Protein G affinity chromatography, followed by a size exclusion chromatographic step. For affinity chromatography supernatant was loaded on a HiTrap Protein A HP column (CV=5 ml, GE Healthcare) coupled to a HiTrap Protein G HP column (CV=5 ml, GE Healthcare) each column equilibrated with 30 ml 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. Unbound protein was removed by washing both columns with 6 column volume 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. Subsequently an additional wash step was necessary to wash only the HiTrap Protein G HP column using at least 8 column volume 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. The target protein was eluted from HiTrap Protein G HP column using a step gradient with 7 column volume 8.8 mM formic acid, pH 3.0. Protein solution was neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8.0. Target protein was concentrated and filtrated prior loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine solution of pH 6.7.

The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of antibodies were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie (SimpleBlue™ SafeStain from Invitrogen). The NuPAGE® Pre-Cast gel system (Invitrogen, USA) was used according to the manufacturer's instruction (4-12% Tris-Acetate gels or 4-12% Bis-Tris). The aggregate content of antibody samples was analyzed using a Superdex 200 10/300GL analytical size-exclusion column (GE Healthcare, Sweden) in 2 mM MOPS, 150 mM NaCl, 0.02% (w/v) NaN3, pH 7.3 running buffer at 25° C.

Analysis of production and purification of an exemplary Fab-Crossfab molecule (consisting of three chains: VHCH1(MCSP)-VLCH1(CD3_(V9))=SEQ ID NO:25, VLCL(MCSP)=SEQ ID NO: 17 and VHCL(CD3_(V9))=SEQ ID NO:23; with an orientation as depicted in FIG. 1 a)) is shown in FIGS. 2 and 3. This molecule is further referred to as Fab (MCSP)-Crossfab (CD3) or hu Fab (MCSP)-Crossfab (CD3).

EXAMPLE 2 Preparation of Fab (MCSP)-Fab (MCSP)-CrossFab(CD3) and Fab (MCSP)-CrossFab(CD3)-Fab (MCSP)

The resulting variable region of heavy and light chain DNA sequences were subcloned in frame with either the constant heavy chain or the constant light chain pre-inserted into the respective recipient mammalian expression vector. The antibody expression is driven by an MPSV promoter and carries a synthetic polyA signal sequence at the 3′ end of the CDS. In addition each vector contains an EBV OriP sequence.

The molecule was produced by co-transfecting HEK293-EBNA cells with the mammalian expression vectors using a calcium phosphate-transfection. Exponentially growing HEK293-EBNA cells were transfected by the calcium phosphate method. Alternatively, HEK293-EBNA cells growing in suspension were transfected by polyethylenimine. The cells were transfected with the corresponding expression vectors in a 1:2:1 ratio (“vector CH1-VH-CH1-VH-CK-VH”:“vector light chain”:“vector light chain CH1-VL”).

For transfection using calcium phosphate cells were grown as adherent monolayer cultures in T-flasks using DMEM culture medium supplemented with 10% (v/v) FCS, and were transfected when they were between 50 and 80% confluent. For the transfection of a T150 flask, 15 million cells were seeded 24 hours before transfection in 25 ml DMEM culture medium supplemented with FCS (at 10% v/v final), and cells were placed at 37° C. in an incubator with a 5% CO2 atmosphere overnight. For each T150 flask to be transfected, a solution of DNA, CaCl2 and water was prepared by mixing 94 μg total plasmid vector DNA divided in the corresponding ratio, water to a final volume of 469 μl and 469 μl of a 1 M CaCl2 solution. To this solution, 938 μl of a 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4 solution at pH 7.05 were added, mixed immediately for 10 s and left to stand at room temperature for 20 s. The suspension was diluted with 10 ml of DMEM supplemented with 2% (v/v) FCS, and added to the T150 in place of the existing medium. Then additional 13 ml of transfection medium were added. The cells were incubated at 37° C., 5% CO2 for about 17 to 20 hours, then medium was replaced with 25 ml DMEM, 10% FCS. The conditioned culture medium was harvested approx. 7 days post-media exchange by centrifugation for 15 min at 210×g, the solution was sterile filtered (0.22 μm filter) and sodium azide in a final concentration of 0.01% (w/v) was added and kept at 4° C. For transfection using polyethylenimine HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 5 min by 210×g, supernatant was replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 μg DNA. After addition of 540 μl PEI solution was vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 (Lonza) was added. After 7 days cultivation supernatant was collected for purification by centrifugation for 15 min at 210×g, the solution was sterile filtered (0.22 μm filter) and sodium azide in a final concentration of 0.01% w/v was added and kept at 4° C.

The secreted protein was purified from cell culture supernatants by affinity chromatography using Protein A and Protein G affinity chromatography, followed by a size exclusion chromatographic step.

For affinity chromatography supernatant was loaded on a HiTrap Protein A HP column (CV=5 ml, GE Healthcare) coupled to a HiTrap Protein G HP column (CV=5 ml, GE Healthcare) each column equilibrated with 30 ml 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. Unbound protein was removed by washing both columns with 6 column volume 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. Subsequently an additional wash step was necessary to wash only the HiTrap Protein G HP column using at least 8 column volume 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. The target protein was eluted from HiTrap Protein G HP column using a step gradient with 7 column volume 8.8 mM formic acid, pH 3.0. Protein solution was neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8.0. Target protein was concentrated and filtrated prior loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine solution of pH 6.7.

The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of antibodies were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie (SimpleBlue™ SafeStain from Invitrogen). The NuPAGE® Pre-Cast gel system (Invitrogen, USA) was used according to the manufacturer's instruction (4-12% Tris-Acetate gels or 4-12% Bis-Tris). The aggregate content of antibody samples was analyzed using a Superdex 200 10/300GL analytical size-exclusion column (GE Healthcare, Sweden) in 2 mM MOPS, 150 mM NaCl, 0.02% (w/v) NaN3, pH 7.3 running buffer at 25° C. and compared with prior art antibody fragment (scFv)2 (results see table below).

Aggregate after 1^(st) Mono- Yield purification HMW LMW mer Construct [mg/l] step [%] [%] [%] [%] (scFv)2 SEQ 3.84 80 0 0 100 ID NOs: 149 Fab-Crossfab 7.85 13.8 0 0 100 SEQ ID NOs: 23, 25, 17 (Fab)2-Crossfab 7.8 3.6 0 0 100 SEQ ID NOs: 42, 17, 23 Fab-Crossfab- 5.3 1.7 0.4 0 99.56 Fab SEQ ID NOs: 27, 17, 23 HMW = High Molecular Weight; LMW = Low Molecular Weight

Analysis of production and purification of an exemplary Fab-Fab-Crossfab molecule (consisting of four chains: VHCH1(MCSP)-VHCH1(MCSP)-VLCH1(CD3_(V9))=SEQ ID NO:26, 2 VLCL(MCSP) chains=SEQ ID NO:17 and one VHCL(CD3_(V9)) chain=SEQ ID NO:23; with an orientation as depicted in FIG. 1 c)) is shown in FIGS. 4 and 5. This molecule is further referred to as Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) or hu Fab (MCSP)-Fab (MCSP)-Crossfab (CD3).

Analysis of production and purification of an exemplary Fab-Crossfab-Fab molecule (consisting of four chains: VHCH1(MCSP)-VLCH1(CD3_(V9))-VHCH1(MCSP)=SEQ ID NO:27, 2 VLCL(MCSP) chains=SEQ ID NO:17 and one VHCL(CD3_(V9)) chain=SEQ ID NO:23; with an orientation as depicted in FIG. 1 e)) is shown in FIGS. 6 and 7. This molecule is further referred to as Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) or hu Fab (MCSP)-Fab (MCSP)-Crossfab (CD3).

Analysis of production and purification of an exemplary Crossfab-Fab-Fab molecule (consisting of four chains: VLCH1(CD3_(2C11))-VHCH1(MCSP)-VHCH1(MCSP)=SEQ ID NO:42, 2 VLCL(MCSP) chains=SEQ ID NO:17 and one VHCL(CD3_(2C11)) chain=SEQ ID NO:43; with an orientation as depicted in FIG. 1 d)) is shown in FIGS. 8 and 9. This molecule is further referred to as murine Crossfab (CD3)-Fab (MCSP)-Fab (MCSP).

EXAMPLE 3 Preparation of Fab(CD33)-CrossFab (CD3)

The resulting variable region of heavy and light chain DNA sequences were subcloned in frame with either the constant heavy chain or the constant light chain pre-inserted into the respective recipient mammalian expression vector. The antibody expression is driven by an MPSV promoter and carries a synthetic polyA signal sequence at the 3′ end of the CDS. In addition each vector contains an EBV OriP sequence.

The molecule was produced by co-transfecting HEK293-EBNA cells with the mammalian expression vectors using a calcium phosphate-transfection. Exponentially growing HEK293-EBNA cells were transfected by the calcium phosphate method. Alternatively, HEK293-EBNA cells growing in suspension were transfected by polyethylenimine. The cells were transfected with the corresponding expression vectors in a 1:1:1 ratio (“vector CH1-VH-CK-VH”:“vector light chain”:“vector light chain CH1-VL”).

For transfection using calcium phosphate cells were grown as adherent monolayer cultures in T-flasks using DMEM culture medium supplemented with 10% (v/v) FCS, and were transfected when they were between 50 and 80% confluent. For the transfection of a T150 flask, 15 million cells were seeded 24 hours before transfection in 25 ml DMEM culture medium supplemented with FCS (at 10% v/v final), and cells were placed at 37° C. in an incubator with a 5% CO2 atmosphere overnight. For each T150 flask to be transfected, a solution of DNA, CaCl2 and water was prepared by mixing 94 μg total plasmid vector DNA divided in the corresponding ratio, water to a final volume of 469 μl and 469 μl of a 1 M CaCl2 solution. To this solution, 938 μl of a 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4 solution at pH 7.05 were added, mixed immediately for 10 s and left to stand at room temperature for 20 s. The suspension was diluted with 10 ml of DMEM supplemented with 2% (v/v) FCS, and added to the T150 in place of the existing medium. Then additional 13 ml of transfection medium were added. The cells were incubated at 37° C., 5% CO2 for about 17 to 20 hours, then medium was replaced with 25 ml DMEM, 10% FCS. The conditioned culture medium was harvested approx. 7 days post-media exchange by centrifugation for 15 min at 210×g, the solution was sterile filtered (0.22 μm filter) and sodium azide in a final concentration of 0.01% (w/v) was added, and kept at 4° C.

For transfection using polyethylenimine HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 5 min by 210×g, supernatant was replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 μg DNA. After addition of 540 μl PEI solution was vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 (LONZA) was added. After 7 days cultivation supernatant was collected for purification by centrifugation for 15 min at 210×g, the solution was sterile filtered (0.22 μm filter) and sodium azide in a final concentration of 0.01% w/v was added, and kept at 4° C.

The secreted protein was purified from cell culture supernatants by affinity chromatography using Protein A and ProteinG affinity chromatography, followed by a size exclusion chromatographic step.

For affinity chromatography supernatant was loaded on a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) coupled to a HiTrap ProteinG HP column (CV=5 mL, GE Healthcare) each column equilibrated with 30 ml 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. Unbound protein was removed by washing both columns with 6 column volume 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. Subsequently an additional wash step was necessary to wash only the HiTrap ProteinG HP column using at least 8 column volume 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. The target protein was eluted from HiTrap ProteinG HP column using a step gradient with 7 column volume 8.8 mM formic acid, pH 3.0. Protein solution was neutralized by adding 1/10 of 0.5M sodium phosphate, pH 8.0. Target protein was concentrated and filtrated prior loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine solution of pH 6.7.

The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of antibodies were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie (SimpleBlue™ SafeStain from Invitrogen). The NuPAGE® Pre-Cast gel system (Invitrogen, USA) was used according to the manufacturer's instruction (4-12% Tris-Acetate gels or 4-12% Bis-Tris). The aggregate content of antibody samples was analyzed using a Superdex 200 10/300GL analytical size-exclusion column (GE Healthcare, Sweden) in 2 mM MOPS, 150 mM NaCl, 0.02% (w/v) NaN3, pH 7.3 running buffer at 25° C.

Analysis of production and purification of an exemplary Fab-Crossfab molecule (consisting of three chains: VHCH1(CD33)-VLCH1(CD3_(V9))=SEQ ID NO:102, VLCL(CD33)=SEQ ID NO:100 and VHCL(CD3_(V9))=SEQ ID NO:23 or SEQ ID NO:101; with an orientation as depicted in FIG. 1 a)) is shown in FIGS. 17 and 18. This molecule is further referred to as Fab(CD33)-CrossFab (CD3) or hu Fab(CD33)-CrossFab (CD3).

EXAMPLE 4 Preparation of the Reference Molecule (scFv)2

Cloning and Production

The resulting variable region of heavy and light chain DNA sequences were subcloned in frame into the respective recipient mammalian expression vector. The antibody expression is driven by an MPSV promoter and carries a synthetic polyA signal sequence at the 3′ end of the CDS. In addition each vector contains an EBV OriP sequence.

The molecule was produced by transfecting HEK293-EBNA cells with the mammalian expression vector using polyethylenimine. HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 5 min by 210×g, supernatant was replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 μg DNA. After addition of 540 μl PEI solution was vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 (LONZA) were added. After 7 days cultivation supernatant was collected for purification by centrifugation for 15 min at 210×g, the solution was sterile filtered (0.22 μm filter) and sodium azide in a final concentration of 0.01% w/v was added, and kept at 4° C.

Purification of (scFv)2 (Anti MCSP/Anti huCD3)

The secreted protein was purified from cell culture supernatants by affinity chromatography using Immobilized Metal Ion Affinity Chromatography (IMAC), followed by a size exclusion chromatographic step.

Prior first purification step disturbing components from the supernatant were removed by diafiltration using the tangential flow filtration system Sarcojet (Sartorius) equipped with a 5.000 MWCO membrane (Sartocon Slice Cassette, Hydrosart; Sartorius). Supernatant was concentrated to 210 ml and subsequently diluted in 1 1 20 mM sodium phosphate, 500 mM sodium chloride, pH 6.5. The protein solution was concentrated again to 210 ml. This process was repeated twice to ensure a complete buffer exchange.

For affinity chromatography retentate of the diafiltration process was loaded on a NiNTA Superflow Cartridge (CV=5 mL, Qiagen) equilibrated with 25 ml 20 mM sodium phosphate, 500 mM sodium chloride, 15 mM imidazole, pH 6.5. Unbound protein was removed by washing with at least 2 column volume 20 mM sodium phosphate, 500 mM sodium chloride, 15 mM imidazole, pH 6.5 followed by an additional wash step using 3 column volume 20 mM sodium phosphate, 500 mM sodium chloride, 62.5 mM imidazole, pH 6.5. Target protein was eluted in 2 column volume 20 mM sodium phosphate, 500 mM sodium chloride, 125 mM imidazole, pH 6.5. Column was washed subsequently with 20 mM sodium phosphate, 500 mM sodium chloride, 250 mM imidazole, pH 6.5.

Target protein was concentrated prior loading on a HiLoad Superdex 75 column (GE Healthcare) equilibrated with 25 mM KH₂PO₄, 125 mM NaCl, 200 mM Arginine, pH 6.7.

Yields, aggregate content after the first purification step and final monomer content is shown in the table above. Comparison of the aggregate content after the first purification step indicates the superior stability of the Fab-Crossfab construct in contrast to the (scFv)2.

Characterization of (scFv)2

The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of antibodies were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie (SimpleBlue™ SafeStain from Invitrogen). The NuPAGE® Pre-Cast gel system (Invitrogen, USA) was used according to the manufacturer's instruction (4-12% Tris-Acetate gels or 4-12% Bis-Tris). The aggregate content of antibody samples was analyzed using a Superdex 75 10/300GL analytical size-exclusion column (GE Healthcare, Sweden) in 2 mM MOPS, 150 mM NaCl, 0.02% (w/v) NaN3, pH 7.3 running buffer at 25° C.

A schematic drawing of the (scFv)2 molecule is shown in FIG. 21.

Analysis of production and purification of an exemplary (scFv)2 molecule (antiMCSP/anti huCD3; consisting two single chain Fvs: VL-VH (MCSP) and VH-VL (CD3_(V9))=SEQ ID NO: 149; is shown in FIGS. 22 and 23. This molecule is further referred to as (scFv)2 (antiMCSP/anti huCD3e).

EXAMPLE 5 Isolation of Primary Human Pan T Cells from PBMCs

Peripheral blood mononuclar cells (PBMCs) were prepared by Histopaque density centrifugation from enriched lymphocyte preparations (buffy coats) obtained from local blood banks or from fresh blood from healthy human donors.

T-cell enrichment from PBMCs was performed using the Pan T Cell Isolation Kit II (Miltenyi Biotec #130-091-156), according to the manufacturer's instructions. Briefly, the cell pellets were diluted in 40 μl cold buffer per 10 Mio cells (PBS with 0.5% BSA, 2 mM EDTA-sterile filtered) and incubated with 10 μl Biotin-Antibody Cocktail per 10 Mio cells for 10 min at 4° C.

30 μl cold buffer and 20 μl Anti-Biotin magnetic beads per 10 Mio cells were added, and the mixture incubated for another 15 min at 4° C.

Cells were washed by adding 10-20× of labeling volume and a subsequent centrifugation step at 300 g for 10 min. Up to 100 Mio cells were resuspended in 500 μl buffer.

Magnetic separation of unlabeled human pan T cells was performed using LS columns (Miltenyi Biotec #130-042-401) according to the manufacturer's instructions. The resulting T cell population was counted automatically (ViCell) and stored in AIM-V medium at 37° C., 5% CO2 in the incubator until assay start (not longer than 24 h).

EXAMPLE 6 Isolation of Murine Pan T Cells from Splenocytes

Spleens were isolated from C57BL/6 mice, transferred into a GentleMACS C-tube (Miltenyi Biotech #130-093-237) containing MACS buffer (PBS+0.5% BSA+2 mM EDTA) and dissociated with the GentleMACS Dissociator to obtain single-cell suspensions according to the manufacturers' instructions.

The cell suspension was passed through a pre-separation filter to get rid-off remaining undissociated tissue particles. After centrifugation at 400 g for 4 minutes at 4° C., ACK Lysis Buffer was added to lyse red blood cells (incubation for 5 minutes at room temperature). The remaining cells were washed with MACS buffer twice, counted and used for the isolation of murine pan T cells. The negative (magnetic) selection was performed using the Pan T Cell Isolation Kit from Miltenyi Biotec (#130-090-861), following the manufacturers' instructions. The resulting T cell population was counted automatically (ViCell) and used immediately for further assays.

EXAMPLE 7 Re-directed T Cell Cytotoxicity Mediated by Cross-linked Bispecific Constructs Targeting CD3 on T Cells and MCSP on Tumor Cells (LDH Release Assay)

Bispecific constructs targeting CD3 on human, or mouse T cells and human on tumor cells, were analyzed by a LDH release assay regarding their potential to induce T cell-mediated apoptosis of target cells.

Briefly, target cells (human Colo-38, human MDA-MB-435, human melanoma MV-3 or murine B16/F10-huMCSP Fluc 2 clone 48 cells, all expressing human MCSP) were harvested with Cell Dissociation Buffer (MCSP is trypsin-sensitive) or trypsin (and then plated the day before), washed and resuspended in the appropriate cell culture medium (see detailed description of the different figures). 20 000-30 000 cells per well were plated in a round-bottom 96-well-plate and the respective antibody dilution was added as indicated (triplicates). Effector cells were added to obtain a final E:T ratio of 5:1 (for human pan T cells), 10:1 (for human PBMCs).

In addition, 1-10 μg/ml PHA-M (Sigma #L8902), a mixture of isolectins, isolated from Phaseolus vulgaris, was used as a mitogenic stimulus to induce human or cynomolgus T cell activation. For murine T cells, a 5% solution of “rat T-Stim with ConA” (BD #354115) was used as a positive control for T cell activation.

For normalization, maximal lysis of the target cells (=100%) was achieved by incubation of the target cells with a final concentration of 1% Triton-X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells, but without any construct or antibody.

After an overnight incubation of at least 18 h at 37° C., 5% CO2, LDH release of apoptotic/necrotic target cells into the supernatant was measured with the LDH detection kit (Roche Applied Science, #11 644 793 001), according to the manufacturer's instructions.

LDH Release Assay with Fab (MCSP)-Crossfab (CD3) and Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) Bispecific Constructs

Purified Fab (MCSP)-Crossfab (CD3), Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) and the (scFv)2 (antiMCSP/anti huCD3e) reference molecule were analyzed for their potential to induce T cell-mediated apoptosis in tumor target cells upon crosslinkage of the construct via binding of both targeting moieties to the respective antigens on cells. Briefly, huMCSP-expressing MDA-MB-435 human melanoma target cells were harvested with Cell Dissociation Buffer, washed and resuspendend in AIM-V medium (Invitrogen #12055-091). 30 000 cells per well were plated in a round-bottom 96-well-plate and the respective antibody dilution was added at the indicated concentrations. All constructs and controls were adjusted to the same molarity.

Human pan T effector cells were added to obtain a final E:T ratio of 5:1. As a positive control for the activation of human pan T cells, 1 μg/ml PHA-M (Sigma #L8902) was used. For normalization, maximal lysis of the target cells (=100%) was determined by incubation of the target cells with a final concentration of 1% Triton-X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells, but without any construct or antibody.

After an overnight incubation of 20 h at 37° C., 5% CO2, LDH release of apoptotic/necrotic target cells into the supernatant was measured with the LDH detection kit (Roche Applied Science, #11 644 793 001), according to the manufacturer's instructions.

As depicted in FIG. 10, the constructs with bivalent MCSP-targeting showed comparable cytotoxic activity compared to the (scFv)2 (antiMCSP/anti huCD3e) construct, whereas the Fab (MCSP)-Crossfab (CD3) construct with monovalent MCSP binding was clearly less potent.

LDH Release Assay with Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) Bispecific Construct with MDA-MB-435 Human Melanoma Target Cells

Purified Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) and the (scFv)2 (antiMCSP/anti huCD3e) reference molecule were analyzed for their potential to induce T cell-mediated apoptosis in tumor target cells upon crosslinkage of the construct via binding of both targeting moieties to the respective antigens on cells.

Briefly, huMCSP-expressing MDA-MB-435 human melanoma target cells were harvested with Cell Dissociation Buffer, washed and resuspendend in AIM-V medium (Invitrogen #12055-091). 30 000 cells per well were plated in a round-bottom 96-well-plate and the respective antibody dilution was added at the indicated concentrations. All constructs and controls were adjusted to the same molarity.

Human pan T effector cells were added to obtain a final E:T ratio of 5:1. As a positive control for the activation of human pan T cells, 5 μg/ml PHA-M (Sigma #L8902) was used. For normalization, maximal lysis of the target cells (=100%) was determined by incubation of the target cells with a final concentration of 1% Triton-X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells, but without any construct or antibody.

After an overnight incubation of 21 h at 37° C., 5% CO2, LDH release of apoptotic/necrotic target cells into the supernatant was measured with the LDH detection kit (Roche Applied Science, #11 644 793 001), according to the manufacturer's instructions.

As depicted in FIG. 11, the Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) induces apoptosis in target cells at least comparably good as the (scFv)2 (antiMCSP/anti huCD3e) molecule.

LDH Release Assay with Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) Bispecific Construct with MV-3 Human Melanoma Target Cells

Purified Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) and the (scFv)2 (antiMCSP/anti huCD3e) molecule were analyzed for their potential to induce T cell-mediated apoptosis in tumor target cells upon crosslinkage of the construct via binding of both targeting moieties to the respective antigens on cells.

Briefly, huMCSP-expressing MV-3 human melanoma target cells were harvested with trypsin on the day before the LDH release assay was started. Cell were washed and resuspendend in the appropriate cell culture medium. 30 000 cells per well were plated in a round-bottom 96-well-plate. The next day, the supernatant was discarded and 100 μl/well AIM-V medium (Invitrogen #12055-091), as well as the respective antibody dilution were added at the indicated concentrations. All constructs and controls were adjusted to the same molarity.

Human PBMC effector cells were added to obtain a final E:T ratio of 10:1. As a positive control for the activation of human pan T cells, 5 μg/ml PHA-M (Sigma #L8902) was used. For normalization, maximal lysis of the target cells (=100%) was determined by incubation of the target cells with a final concentration of 1% Triton-X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells, but without any construct or antibody.

After an overnight incubation of 26 h at 37° C., 5% CO2, LDH release of apoptotic/necrotic target cells into the supernatant was measured with the LDH detection kit (Roche Applied Science, #11 644 793 001), according to the manufacturer's instructions.

As depicted in FIG. 12, the Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) induces apoptosis in target cells at least comparably good as the (scFv)2 (antiMCSP/anti huCD3e) molecule.

LDH Release Assay with Fab (MCSP)-Crossfab (CD3) Bispecific Construct with MV-3 Human Melanoma Target Cells

An LDH release assay was performed as outlined above. FIG. 19 shows killing of huMCSP-positive MV-3 tumor cells upon co-culture with human PBMCs (E:T ratio=10:1), treated with Fab (MCSP)-Crossfab (CD3), respective the (scFv)2 (antiMCSP/anti huCD3e) reference molecule for ˜24 hours.

LDH Release Assay with Murine Crossfab (CD3)-Fab (MCSP)-Fab (MCSP) Bispecific Construct

Purified with murine Crossfab (CD3)-Fab (MCSP)-Fab (MCSP), targeting murine CD3, as well as human MCSP, was analyzed for its potential to induce T cell-mediated apoptosis in tumor target cells upon crosslinkage of the construct via binding of both targeting moieties to the respective antigens on cells.

Briefly, huMCSP-expressing B16/F10-huMCSP Fluc2 clone 48 tumor target cells were harvested with Cell Dissociation Buffer, washed and resuspendend in RPMI1640 medium, including 1×NEAA, 10 mM Hepes, 50 μm 2-b-ME and 1 mM sodium pyruvate.

20 000 cells per well were plated in a round-bottom 96-well-plate and the respective antibody dilution was added at the indicated concentrations. The bispecific construct and the different IgG controls were adjusted to the same molarity. As an additional control for the activation of murine T cells “T Cell Stim with ConA” (BD #354115) was used, diluted 1:160 with assay medium.

Murine pan T effector cells, isolated from splenocytes (C57BL/6 mice) were added to obtain a final E:T ratio of 10:1. For normalization, maximal lysis of the target cells (=100%) was determined by incubation of the target cells with a final concentration of 1% Triton-X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells, but without any construct or antibody.

After an incubation for 70 h at 37° C., 5% CO2, LDH release of apoptotic/necrotic target cells into the supernatant was measured with the LDH detection kit (Roche Applied Science, #11 644 793 001), according to the manufacturer's instructions.

As depicted in FIG. 13, the bispecific construct induces concentration-dependent LDH release from target cells, comparable to the positive control with “T Cell Stim with ConA”.

LDH Release Assay with Murine Crossfab (CD3)-Fab (MCSP)-Fab (MCSP) Bispecific Construct

Purified murine Crossfab (CD3)-Fab (MCSP)-Fab (MCSP), targeting murine CD3, as well as human MCSP, was analyzed for its potential to induce T cell-mediated apoptosis in tumor target cells upon crosslinkage of the construct via binding of both targeting moieties to the respective antigens on cells.

Briefly, huMCSP-expressing B16/F10-huMCSP Fluc2 clone 48 tumor target cells were harvested with Cell Dissociation Buffer, washed and resuspendend in RPMI1640 medium, including 1×NEAA, 10 mM Hepes, 50 μM 2-b-ME and 1 mM sodium pyruvate.

20 000 cells per well were plated in a round-bottom 96-well-plate and the respective antibody dilution was added to obtain a final concentration of 50 nM. The bispecific construct and the different IgG controls were adjusted to the same molarity.

Murine pan T effector cells, isolated from splenocytes (C57BL/6 mice) were added to obtain a final E:T ratio of 10:1. To assess the level of hyperactivation of murine T cells in the absence of target cells, control wells with 50 nM bispecific construct and T cells were plated accordingly.

For normalization, maximal lysis of the target cells (=100%) was determined by incubation of the target cells with a final concentration of 1% Triton-X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells, but without any construct or antibody.

After an incubation for 70 h at 37° C., 5% CO2, LDH release of apoptotic/necrotic target cells into the supernatant was measured with the LDH detection kit (Roche Applied Science, #11 644 793 001), according to the manufacturer's instructions.

As depicted in FIG. 14, the bispecific construct induces strong LDH release from target cells. In the absence of target cells, there was only a slight increase of LDH (reflecting hyperactivation of T cells) compared to untreated murine T cells, co-incubated with target cells. None of the control IgGs induces LDH release of target cells.

EXAMPLE 8 Cytokine Release Assay (CBA Analysis)

To assess the de novo secretion of different cytokines upon T cell activation with CD3-bispecific constructs in the presence or absence of target cells, human PBMCs were isolated from Buffy Coats and 0.3 Mio cells per well were plated into a round-bottom 96-well plate. Alternatively, 280 μl whole blood from a healthy donor were plated per well of a deep-well 96-well plate.

Tumor target cells (e.g. MDA-MB-435 cells for CD3-MCSP-bispecific constructs) were added to obtain a final E/T-ratio of 10:1. Bispecific constructs and controls were added as indicated. After an incubation of up to 24 h at 37° C., 5% CO2, the assay plate was centrifuged for 5 min at 350 g and the supernatant was transferred into a new deep-well 96-well-plate for the subsequent analysis.

The CBA analysis was performed according to manufacturers' instructions for FACS CantoII, using the combination of the following CBA Flex Sets: human granzyme B (BD 560304), human IFN-γ Flex Set (BD 558269), human TNF Flex Set (BD 558273), human IL-10 Flex Set (BD 558274), human IL-6 Flex Set (BD 558276), human IL-4 Flex Set (BD 558272).

Cytokine Release Assay with MCSP-CD3 Bispecific Constructs

The following purified bispecific constructs targeting human MCSP and human CD3 were analyzed for their ability to induce T cell-mediated de novo secretion of cytokines in the presence (A, B) versus absence (C, D) of tumor target cells: “Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) and the (scFv)2 (antiMCSP/anti huCD3e) reference molecule.

Briefly, 280 μl whole blood from a healthy donor were plated per well of a deep-well 96-well plate. 30 000 Colo-38 tumor target cells, expressing human MCSP, as well as the different bispecific constructs and IgG controls were added were added at 1 nM final concentration. The cells were incubated for 24 h at 37° C., 5% CO2 and then centrifuged for 5 min at 350×g. The supernatant was transferred into a new deep-well 96-well-plate for the subsequent analysis.

The CBA analysis was performed according to manufacturers' instructions for FACS CantoII, using the combination of the following CBA Flex Sets: human granzyme B (BD 560304), human IFN-γ Flex Set (BD 558269), human TNF Flex Set (BD 558273), human IL-10 Flex Set (BD 558274), human IL-6 Flex Set (BD 558276), human IL-4 Flex Set (BD 558272).

FIG. 15 depicts different cytokine levels, that were measured in the supernatant of whole blood after treatment with 1 nM of different CD3-MCSP bispecific constructs (Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) and the (scFv)2 (antiMCSP/anti huCD3e)) in the presence (A, B) or absence (C,D) of Colo-38 tumor cells for 24 hours. 280 μl whole blood were plated per well of a 96-well plate and 30 000 Colo-38 cells added, as indicated.

The main cytokine that was secreted upon activation of T cells in the presence of Colo-38 tumor cells, was IL-6, followed by IFNgamma. In addition, also the levels of granzyme B increased enormously upon activation of T cells in the presence of target cells. In general, the (scFv)2 (antiMCSP/anti huCD3e) construct elevated the levels of TNF and IFNgamma, as well as granzyme B in the presence of target cells (A and B) a bit more compared to the other bispecific construct.

There was no significant secretion of Th2 cytokines (IL-10 and IL-4) upon activation of T cells by the bispecific constructs in the presence (or absence) of target cells.

In this assay, there was also a weak secretion of IFNgamma, induced by the Fab (MCSP)-Fab (MCSP)-Crossfab (CD3) construct in the absence of target cells.

Cytokine Release Assay with MCSP-murineCD3 Bispecific Constructs

The purified huMCSP-muCD3-targeting bispecific molecule as murine Crossfab (CD3)-Fab (MCSP)-Fab (MCSP) was tested by flow cytometry for its potential to up-regulate the late activation marker CD25 on CD8+ T cells in the presence of human MCSP-expressing tumor cells.

Briefly, MCSP-positive B16/F10-huMCSP Fluc2 clone 48 tumor cells were harvested with Cell Dissociation buffer, counted and checked for viability. Cells were adjusted to 0.3×10⁶ (viable) cells per ml in RPMI1640 medium (including 1×NEAA, 10 mM Hepes, 50 μm 2-b-ME, 1 mM sodium pyruvate), 100 μl of this cell suspension were pipetted per well into a round-bottom 96-well plate (as indicated). 50 μl of the (diluted) bispecific construct was added to the cell-containing wells to obtain a final concentration of 50 nM. Human murine T effector cells were isolated from splenocytes (C57BL/6 mice) and adjusted to 3×10⁶ (viable) cells per ml in AIM-V medium. 50 μl of this cell suspension was added per well of the assay plate (see above) to obtain a final E:T ratio of 10:1. To analyze, if the bispecific construct was able to activate T cells only in the presence of target cells, expressing huMCSP, wells were included that contained 50 nM of the respective bispecific molecule, as well as T effector, but no target cells.

After incubation for 70 hours at 37° C., 5% CO2, cells were centrifuged (5 min, 350×g) and washed twice with 150 l/well PBS, including 0.1% BSA.

Surface staining for CD8a (rat IgG2a; clone 53-6.7; BioLegend #100712) and CD25 (rat IgG2b; clone 3C7; BD #553075) was performed according to the suppliers' suggestions. Cells were washed twice with 150 l/well PBS, including 0.1% BSA and fixed for 15 min at 4° C., using 100 l/well fixation buffer (BD ##554655).

After centrifugation, the samples were resuspended in 200 μl/well PBS, 0.1% BSA and analyzed using a FACS CantoII machine (Software FACS Diva).

FIG. 16 shows that the as murine Crossfab (CD3)-Fab (MCSP)-Fab (MCSP) construct induces up-regulation of CD25 in the presence of target cells only.

EXAMPLE 9 Expression of Surface Activation Markers on Primary Human T Cells Upon Engagement of Bispecific Constructs

To check for specific activation of T cells upon binding of CD3 bispecific constructs exclusively in the presence of tumor target cells, primary human PBMCs (isolated as described above) were incubated with the indicated concentrations of bispecific constructs for at least 24 h in the presence or absence of tumor antigen-positive target cells.

Briefly, 0.3 million primary human PBMCs were plated per well of a flat-bottom 96-well plate, containing the huMCSP-positive target cells (MV-3 tumor cells) or medium. The final effector to target cell (E:T) ratio was 10:1. The cells were incubated with the indicated concentration of the CD3-MCSP bispecific constructs (Fab (MCSP)-Crossfab (CD3); designated as “1+1 non-Fc”, and the (scFv)2 (antiMCSP/anti huCD3e) reference molecule (designated as “(scFv)2”) for the indicated incubation times at 37° C., 5% CO2. The effector cells were stained for CD8, and the early activation marker CD69 or the late activation marker CD25 and analyzed by FACS CantoII.

FIG. 20 shows the result of this experiment.

EXAMPLE 10 Preparation of Fab-CrossFab Linked Light Chain (Fab-Crossfab with Two Linkers)

The molecule was produced by co-transfecting HEK293-EBNA cells growing in suspension with the mammalian expression vectors using by polyethylenimine. The cells were transfected with the corresponding expression vectors in a 1:1 ratio (“vector heavy chain” “vector linked light chain”).

HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 5 min by 210×g, supernatant was replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 μg DNA. After addition of 540 μl PEI solution was vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 was added. After 7 days cultivation supernatant was collected for purification by centrifugation for 15 min at 210×g, the solution was sterile filtered (0.22 μm filter) and sodium azide in a final concentration of 0.01% w/v was added, and kept at 4° C.

The secreted protein was purified from cell culture supernatants by affinity chromatography using ProteinG affinity chromatography, followed by a size exclusion chromatographic step.

For affinity chromatography supernatant was loaded on a HiTrap ProteinG HP column (CV=5 mL, GE Healthcare) equilibrated with 30 ml 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. Unbound protein was removed by washing both columns with 6 column volume 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. Subsequently column was washed using at least 8 column volume 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. The target protein was eluted from HiTrap ProteinG HP column using a step gradient with 7 column volume 8.8 mM formic acid, pH 3.0. Protein solution was directly loaded on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine solution of pH 6.7.

The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.

Purity and molecular weight of the construct was analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie (SimpleBlue™ SafeStain from Invitrogen). The NuPAGE® Pre-Cast gel system (Invitrogen, USA) was used according to the manufacturer's instruction (4-12% Bis-Tris).

The aggregate content of antibody samples was analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25° C. Final monomer content was 99.4%.

Aggregate after 1^(st) Mono- Construct Yield purification HMW LMW mer MCSP [mg/l] step [%] [%] [%] [%] 1 + 1 Fab-Cross- 0.67 27.0 0.6 0 99.4 fab linked light chain (hu Fab (MCSP) = Cross- fab (CD3)) SEQ ID NOs 163 and 164

Analysis of production and purification of an exemplary Fab-Crossfab molecule with two linkers (consisting of two chains: VHCH1(MCSP)-(G4S)2-VLCH1(CD3_(V9))=SEQ ID NO: 164, VLCL(MCSP)-(G4S)2-VHCL(CD3_(V9))=SEQ ID NO: 163; with an orientation as depicted in FIG. 24 a) is shown in FIG. 25. This molecule is further referred to as Fab (MCSP)=Crossfab (CD3) or hu Fab (MCSP)=Crossfab (CD3).

Thermal Stability of Fab-CrossFab Linked Light Chain

Thermal stability of the protein was monitored by Dynamic Light Scattering (DLS). 30 μg of filtered protein sample with a protein concentration of 1 mg/ml was applied in duplicate to a Dynapro plate reader (Wyatt Technology Corporation; USA). The temperature was ramped from 25 to 75° C. at 0.05° C./min, with the radius and total scattering intensity being collected.

The results are shown in FIG. 28. Thermal stability was analyzed for the an exemplary Fab-Crossfab molecule with two linkers (consisting of two chains: VHCH1(MCSP)-(G4S)2-VLCH1(CD3_(V9))=SEQ ID NO: 164, VLCL(MCSP)-(G4S)2-VHCL(CD3_(V9))=SEQ ID NO: 163; with an orientation as depicted in FIG. 24 a) in comparison to the (scFv)2 molecule. The aggregation temperature of the Fab-CrossFab linked light chain molecule was measured at 63° C. (black line). The (scFv)2 molecule was inferior to the Fab-Crossfab molecule with two linkers since it starts to aggregate already at 45° C. (grey line).

The Fab-Crossfab molecule with two linkers has a higher stability and thus more developable than the (scFv)2 molecule.

EXAMPLE 11 Analysis of Fab-Crossfab with Two Linkers (Fab (MCSP)=Crossfab (CD3))

Isolation of Primary Human PBMCs from Fresh Blood or Buffy Coat

Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation from enriched lymphocyte preparations (buffy coats) obtained from local blood banks or from fresh blood from healthy human donors. Briefly, blood was diluted with sterile PBS and carefully layered over a Histopaque gradient (Sigma, H8889). After centrifugation for 30 minutes at 450×g at room temperature (brake switched-off), part of the plasma above the PBMC containing interphase was discarded. Transfer the PBMCs into new 50 ml falcon tubes and fill-up the tubes with PBS to 50 ml total volume. Switch-on the brake and centrifuge at room temperature for 10 minutes at 400×g. Discard the supernatant and wash the PBMC pellet twice with sterile PBS (centrifugation steps at 4° C. for 10 minutes at 350×g).

The resulting PBMC population was counted automatically (ViCell) and stored in RPMI1640 medium, containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37° C., 5% CO₂ in the incubator until assay start.

FACS Analysis of Surface Activation Markers on Primary Human T Cells Upon Engagement of Bispecific Constructs

To check for specific activation of T cells upon binding of CD3 bispecific constructs exclusively in the presence of tumor target cells, primary human PBMCs were incubated with the indicated concentrations of bispecific constructs for at least 22 h in the presence or absence of MCSP-positive target cells. Briefly, 0.3 million primary human PBMCs were plated per well of a flat-bottom 96-well plate, containing the MCSP-positive target cells (or medium). The final effector to target cell (E:T) ratio was 10:1. The cells were incubated with the indicated concentration of the bispecific constructs and controls for the indicated incubation times at 37° C., 5% CO₂. The effector cells were stained for CD8, and the early activation marker CD69 or the late activation marker CD25 and analyzed by FACS CantoII.

FIG. 27 shows the results of this assay: Activation of CD4+ and CD8+ T cells, mediated by different CD3-MCSP bispecific constructs in the presence versus absence of MCSP-positive MV-3 tumor cells after 24 hours (E:T=10:1), measured as up-regulation of CD69 median fluorescence (A), respective percent of CD69 positive cells (B).

LDH Release Assay

Bispecific constructs targeting CD3 on human T cells, and human MCSP on tumor cells were analyzed by a LDH release assay for their potential to induce T cell-mediated apoptosis of target cells. Briefly, target cells (human Colo-38 or human MV-3 melanoma cells) were harvested with Cell Dissociation Buffer on the day of the assay (or with trypsin one day before the assay was started), washed and resuspended in the appropriate cell culture medium (as indicated). 20 000-30 000 cells per well were plated in a flat-bottom 96-well plate and the respective antibody dilution was added as indicated (triplicates). Effector cells were added to obtain a final E:T ratio of 10:1. All constructs and controls were adjusted to the same molarity. For normalization, maximal lysis of the target cells (=100%) was achieved by incubation of the target cells with a final concentration of 1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells, but without any bispecific construct. After an overnight incubation of at least 22 h at 37° C., 5% CO₂, LDH release of apoptotic/necrotic target cells into the supernatant was measured, using the LDH detection kit (Roche Applied Science, #11 644 793 001), according to the manufacturer's instructions.

FIG. 26 shows the results of this assay: Killing (as measured by LDH release) of MCSP-positive MV-3 tumor cells upon co-culture with human PBMCs (E:T ratio=10:1), treated with different CD3-MCSP bispecific constructs for ˜24 hours.

EC50 values were determined using Graph Pad Prism software.

hu Fab (MCSP)- hu Fab (MCSP) = Crossfab (CD3) Crossfab (CD3) (1 linker) (2 linkers) (scFv)2 SEQ ID NO: SEQ ID NOs: SEQ ID NO: 25, 23, 17 163, 164 149 EC50 (pM) 25.35 89.78 57.1

While there are shown and described presently preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

EXAMPLE 12 Affinity Maturation of Anti-MCSP Antibody M4-3/ML2

The affinity maturation is described in European patent application EP 13156686.1, which is incorporated by reference in its entirety.

Affinity maturation was performed via the oligonucleotide-directed mutagenesis procedure. For this the heavy chain variant M4-3, and the light chain variant ML2 were cloned into a phagemid vector, similar to those described by Hoogenboom, (Hoogenboom et al., Nucleic Acids Res. 1991, 19, 4133-4137). Residues to be randomized were identified by first generating a 3D model of that antibody via classical homology modeling and then identifying the solvent accessible residues of the complementary determining regions (CDRs) of heavy and light chain. Oligonucleotides with randomization based on trinucleotide synthesis as shown in table 3 were purchased from Ella-biotech (Munich, Germany). Three independent sublibraries were generated via classical PCR, and comprised randomization in CDR-H1 together with CDR-H2, or CDR-L1 together with CDR-L2, CDR-L3 was randomized in a separate approach. The DNA fragments of those libraries were cloned into the phagemid via restriction digest and ligation, and subsequently electroporated into TG1 bacteria.

Library Selection.

The antibody variants thus generated were displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants were then screened for their biological activities (here: binding affinity) and candidates that have one or more improved activities were used for further development. Methods for making phage display libraries can be found in Lee et al., J. Mol. Biol. (2004) 340, 1073-1093),

Selections with all affinity maturation libraries were carried out in solution according to the following procedure: 1. binding of˜1012 phagemid particles of each affinity maturation libraries to 100 nM biotinylated hu-MCSP(D3 domain)-avi-his(SEQ ID NO. 118) for 0.5 h in a total volume of 1 ml, 2. capture of biotinylated hu-MCSP(D3 domain)-avi-his and specifically bound phage particles by addition of 5.4×107 streptavidin-coated magnetic beads for 10 min, 3. washing of beads using 5-10x 1 ml PBS/Tween20™ and 5-10x 1 ml PBS, 4. elution of phage particles by addition of 1 ml 100 mM TEA (triethylamine) for 10 min and neutralization by adding 500 ul 1 M Tris/HCl pH 7.4 and 5. re-infection of exponentially growing E. coli TG1 bacteria, infection with helper phage VCSM13 and subsequent PEG/NaCl precipitation of phagemid particles to be used in subsequent selection rounds. Selections were carried out over 3-5 rounds using either constant or decreasing (from 10-7 M to 2×10-9 M) antigen concentrations. In round 2, capture of antigen: phage complexes was performed using neutravidin plates instead of streptavidin beads. Specific binders were identified by ELISA as follows: 100 ul of 10 nM biotinylated hu-MCSP(D3 domain)-avi-his per well were coated on neutravidin plates. Fab-containing bacterial supernatants were added and binding Fabs were detected via their Flag-tags by using an anti-Flag/HRP secondary antibody. ELISA-positive clones were bacterially expressed as soluble Fab fragments in 96-well format and supernatants were subjected to a kinetic screening experiment by SPR-analysis using PROTEON™ XPR36 (BioRad). Clones expressing Fabs with the highest affinity constants were identified and the corresponding phagemids were sequenced.

TABLE 3 (excluded were always Cys, and Met. Lys was excluded on top in those cases where the oligonucleotide was a reverse primer) Position Randomization Heavy chain CDR1 Ser31 S (40%), test (60%, 4% each) Gly32 G (40%), rest (60%, 4% each). Tyr33 Y (40%), rest (60%, 4% each) Tyr34 Y (40%), rest (60%, 4% each) CDR2 Tyr50 Y 40%, (F, W, L, A, I, 30%, 6% each), rest (30%, 2.5% each) Thr52 T (60%), rest (40%, 2.5% each) Tyr53 Y (40%), rest (60%, 3.8% each) Asp54 D (40%), rest (60%, 3.8% each) Ser56 S (40%), rest (60%, 3.8% each) Light chain CDR1 Gln27 Q (40%), (E, D, N, S, T, R, 40%, 6.7% each), rest (total 20%, 2.2% each) Gly28 G (40%), (N, T, S, Q, Y, D, E, 40%, 5.7% each), rest (20%, 2.5% each) Asn31 N (40%), (S, T, G, Q, Y, D, E, R, 50%, 6.3% each), rest (10%, 1.4% each) Tyr32 Y (40%), (W, S, R, 30%, 10% each), rest (30%, 2.3% each) CDR2 Tyr50 Y (70%), (E, R, K, A, Q, T, S, D, G, W, F, 30%, 2.7% each) Thr51 T (50%), (S, A, G, N, Q, V, 30%, 5% each), rest (20%, 2% each) Ser52 S (50%), rest (50%, 3.1% each) Ser53 S (40%), (N, T, Q, Y, D, E, I, 40%, 5.7% each), rest (20%, 2.2% each) CDR3 Tyr91 Y (50%), rest (50%, 3.1% each) Ser92 S (50%), (N, Q, T, A, G 25%, 5% each), rest (25%, 2.3% each) Lys93 K (50%), S (5%), T (5%), N (5%), rest (35%, 2.7% each) Leu94 L (50%), (Y, F, S, I, A, V, 30%, 5% each), rest (20%, 2% each) Pro95 P (50%), (S, A, 20%, 10% each), rest (30%, 2.1% each) Trp96 W 50%, (Y, R, L, 15%, 5% each), rest (35%, 2.5% each)

Heavy chain randomization was performed only in the CDR1 and 2. Light chain randomization was performed in CDR1 and 2, and independently in CDR3.

During selection, a few mutations in the frameworks occured like F71Y in clone G3 or Y87H in clone E10.

Production and Purification of Human IgG1

The variable region of heavy and light chain DNA sequences of the affinity matured variants were subcloned in frame with either the constant heavy chain or the constant light chain pre-inserted into the respective recipient mammalian expression vector. The antibody expression was driven by an MPSV promoter and carries a synthetic polyA signal sequence at the 3′ end of the CDS. In addition each vector contained an EBV OriP sequence.

The molecule was produced by co-transfecting HEK293-EBNA cells with the mammalian expression vectors using polyethylenimine. The cells were transfected with the corresponding expression vectors in a 1:1 ratio. For transfection HEK293 EBNA cells were cultivated in suspension serum free in CD CHO culture medium. For the production in 500 ml shake flask 400 million HEK293 EBNA cells were seeded 24 hours before transfection. For transfection cells were centrifuged for 5 min by 210×g, supernatant was replaced by pre-warmed 20 ml CD CHO medium. Expression vectors were mixed in 20 ml CD CHO medium to a final amount of 200 μg DNA. After addition of 540 μ1 PEI solution was vortexed for 15 s and subsequently incubated for 10 min at room temperature. Afterwards cells were mixed with the DNA/PEI solution, transferred to a 500 ml shake flask and incubated for 3 hours by 37° C. in an incubator with a 5% CO2 atmosphere. After incubation time 160 ml F17 medium was added and cell were cultivated for 24 hours. One day after transfection 1 mM valporic acid and 7% Feed 1 was added. After 7 days cultivation supernatant was collected for purification by centrifugation for 15 min at 210×g, the solution was sterile filtered (0.22 μm filter) and sodium azide in a final concentration of 0.01% w/v was added, and kept at 4° C.

The secreted protein was purified from cell culture supernatants by affinity chromatography using ProteinA. Supernatant was loaded on a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare) equilibrated with 40 ml 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. Unbound protein was removed by washing with at least 10 column volume 20 mM sodium phosphate, 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5. Target protein was eluted during a gradient over 20 column volume from 20 mM sodium citrate, 0.5 M sodium chloride, pH 7.5 to 20 mM sodium citrate, 0.5 M sodium chloride, pH 2.5. Protein solution was neutralized by adding 1/10 of 0.5 M sodium phosphate, pH 8. Target protein was concentrated and filtrated prior loading on a HiLoad Superdex 200 column (GE Healthcare) equilibrated with 20 mM Histidine, 140 mM sodium chloride solution of pH 6.0.

The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and molecular weight of molecules were analyzed by CE-SDS analyses in the presence and absence of a reducing agent. The Caliper LABCHIP® GXII system (Caliper lifescience) was used according to the manufacturer's instruction. 2ug sample is used for analyses. The aggregate content of antibody samples is analyzed using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at 25° C.

Affinity Determination

ProteOn Analysis

KD was measured by surface plasmon resonance using a PROTEON™ XPR36 machine (BioRad) at 25° C. with anti-human F(ab')2 fragment specific capture antibody (Jackson ImmunoResearch #109-005-006) immobilized by amine coupling on CM5 chips and subsequent capture of Fabs from bacterial supernatant or from purified Fab preparations. Briefly, carboxymethylated dextran biosensor chips (CM5, GE Healthcare) were activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Anti-human F(ab')2 fragment specific capture antibody was diluted with 10 mM sodium acetate, pH 5.0 at 50 μg/ml before injection at a flow rate of 10 μl/minute to achieve approximately up to 10.000 response units (RU) of coupled capture antibody. Following the injection of the capture antibody, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, Fabs from bacterial supernatant or purified Fabs were injected at a flow rate of 10 ul/minute for 300s and a dissociation of 300s for capture baseline stabilization. Capture levels were in the range of 100-500 RU. In a subsequent step, human MCSP(D3 domain)-avi-his analyte was injected either as a single concentration or as a concentration series (depending of clone affinity in a range between 100 nM and 250 pM) diluted into HBS-EP+ (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20, pH 7.4) at 25° C. at a flow rate of 50 μl/min. The surface of the sensorchip was regenerated by injection of glycine pH 1.5 for 30s at 90 ul/min followed by injection of NaOH for 20s at the same flow rate. Association rates (kon) and dissociation rates (koff) were calculated using a simple one-to-one Langmuir binding model (PROTEON™ XPR36 Evaluation Software or Scrubber software (BioLogic)) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) was calculated as the ratiokoff/kon. . This data was used to determine the comparative binding affinity of the affinity matured variants with the parental antibody. Table 4a shows the data generated from these assays.

G3, E10, C5 for the Light Chain, and D6, A7, B7, B8, C1 for the Heavy Chain were chosen for conversion into human IgG1 format. Since CDR1 and 2 of the light chain were randomized independent from CDR3, the obtained CDRs were combined during IgG conversion.

In the IgG format affinities were measured again to the human MCSP antigen in addition also to the cynomolgus homologue.

Method exactly as described for the Fab fragments, just purified IgG from mammalian production were used.

TABLE 4a MCSP acuity matured clones: Proteon data Human Cyno MCSP MCSP Human Human Cyno IgG KD IgG KD MCSP MCSP MCSP Comparative Fab KD IgG KD IgG KD binding affinity - Proteon generated Fold increase Variant affinity data over parent Parental M4-3/ML2 5*10⁻⁹  2*10⁻⁹  2*10⁻⁹  M4-3/ML2(G3) 4*10⁻¹⁰ 3*10⁻¹⁰ 6*10⁻¹⁰ 6.7 3.3 M4-3/ML2 (E10) 7*10⁻¹⁰ 1*10⁻⁹  2*10⁻⁹  2.0 1.0 M4-3/ML2 (E10/G3) 4*10⁻¹⁰ 9*10⁻¹⁰ 5.0 2.2 M4-3/ML2 (C5) 7*10⁻¹⁰ 4*10⁻¹⁰ 1*10⁻⁹  5.0 2.0 M4-3/ML2 (C5/G3) 7*10⁻¹⁰ 1*10⁻⁹  2.9 2.0 M4-3(D6)/ML2 2*10⁻⁹  4*10⁻¹⁰ 1*10⁻⁹  5.0 2.0 M4-3(A7)/ML2 2*10⁻¹¹ 8*10⁻¹⁰ 1*10⁻⁹  2.5 2.0 M4-3(B7)/ML2 5*10⁻¹⁰ 7*10⁻¹⁰ 4.0 2.9 M4-3(B8)/ML2 3*10⁻¹⁰ 9*10⁻¹⁰ 1*10⁻⁹  2.2 2.0 M4-3(C1)/ML2 6*10⁻¹⁰ 9*10⁻¹⁰ 8*10⁻¹⁰ 2.2 2.5 M4-3(C1)/ML2(G3) 7*10⁻¹¹ 2*10⁻¹⁰ 28.6 10.0 M4-3(C1)/ML2(E10) 5*10⁻¹⁰ 6*10⁻¹⁰ 4.0 3.3 M4-3(A7)/ML2(G3) 7*10⁻¹¹ 2*10⁻¹⁰ 28.6 10.0 M4-3(A7)/ML2(E10) 3*10⁻¹⁰ 7*10⁻¹⁰ 6.7 2.9 M4-3(C1)/ML2(C5) 2*10⁻¹⁰ 3*10⁻¹⁰ 10.0 6.7 M4-3(A7)/ML2(C5) 7*10⁻¹¹ 2*10⁻¹⁰ 28.6 10.0 Affinity Determination by Surface Plasmon Resonance (SPR) Using Biacore T200

Surface plasmon resonance (SPR) experiments to determine the affinity and the avidity of the affinity matured IgGs were performed on a BIACORE® T200 at 25° C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).

For analyzing the avidity of the interaction of different antiMCSP IgGs to human and cynomolgus MCSP D3 direct coupling of around 9,500 resonance units (RU) of the anti-Penta His antibody (Qiagen) (“PentaHis” disclosed as SEQ ID NO: 208) was performed on a CM5 chip at pH 5.0 using the standard amine coupling kit (Biacore, Freiburg/Germany). Antigens were captured for 60 s at 30 nM with 10 μl/min respectively. IgGs were passed at a concentration of 0.0064-100 nM with a flowrate of 30 μl/min through the flow cells over 280 s. The dissociation was monitored for 180 s. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell. Here, the IgGs were flown over a surface with immobilized anti-Penta His antibody (“PentaHis” disclosed as SEQ ID NO: 208) but on which HBS-EP has been injected rather than human MCSP D3 or cynomolgus MCSP D3.

For affinity measurements IgGs were captured on a CM5 sensorchip surface with immobilized anti human Fc. Capture IgG was coupled to the sensorchip surface by direct immobilization of around 9,500 resonance units (RU) at pH 5.0 using the standard amine coupling kit (Biacore, Freiburg/Germany). IgGs are captured for 25 s at 10 nM with 30 μl/min. Human and cynomolgus MCSP D3 were passed at a concentration of 2-500 nM with a flowrate of 30 μl/min through the flow cells over 120 s. The dissociation is monitored for 60 s. Association and dissociation for concentration 166 and 500 nM was monitored for 1200 and 600 s respectively. Bulk refractive index differences were corrected for by subtracting the response obtained on reference flow cell. Here, the antigens were flown over a surface with immobilized anti-human Fc antibody but on which HBS-EP has been injected rather than anti MCSP IgGs.

Kinetic constants were derived using the BIACORE® T200 Evaluation Software (vAA, Biacore AB, Uppsala/Sweden), to fit rate equations for 1:1 Langmuir binding by numerical integration.

Higher affinity to human and cynomolgus MCSP D3 were confirmed by surface plasmon resonance measurements using BIACORE® T200. In addition avidity measurements showed an up to 3fold increase in bivalent binding (Table 4b).

TABLE 4b Affinity and avidity of anti MCSP IgGs to human MCSP-D3 and cynomolgus MCSP D3. K_(D) in nM Human MCSP D3 Cynomolgus MCSP D3 T = 25° C. Affinity Avidity Affinity Avidity M4-3(C1) ML2(G3) 1.8 0.0045 1.4 0.0038 M4-3(C1) ML2(E10) 4.6 0.0063 3.8 0.0044 M4-3(C1) ML2(C5) 1.8 0.0046 1.3 0.0044 M4-3 ML2 (parental) 8.6 0.0090 11.4 0.0123

Sequences

Legend: GA201=EGFR binder, 3F2=FAP binder, CH1A1A=CEA binder.

Protein Sequences

SEQ ID. NO. Description Sequence 1 CDR1 VL MCSP SASQGIRNYLN 2 CDR2 VL MCSP YTSSLHS 3 CDR3 VL MCSP QQYSKLPWT 4 CDR1 VH MCSP GYSITSGYYWN 5 CDR2 VH MCSP YITYDGSNNYNPSLKN 6 CDR3 VH MCSP FDY 7 CDR1 VL CD3_((V9)) RASQDIRNYLN 8 CDR2 VL CD3_((V9)) YTSRLES 9 CDR3 VL CD3_((V9)) QQGNTLPWT 10 CDR1 VH CD3_((V9)) GYTMN 11 CDR2 VH CD3_((V9)) LINPYKGVSTYNQKFKD 12 CDR3 VH CD3_((V9)) SGYYGDSDWYFDV 29 CDR1 VL CD3_((H2C)) GSSTGAVTSGYYPN 30 CDR2 VL CD3_((H2C)) GTKFLAP 31 CDR3 VL CD3_((H2C)) ALWYSNRWV 32 CDR1 VH CD3_((H2C)) GFTFNKYAMN 33 CDR2 VH CD3_((H2C)) RIRSKYNNYATYYADSVKD 34 CDR3 VH CD3_((H2C)) HGNFGNSYISYWAY 13 VL MCSP DIVLTQSPSSLSASLGDRVTISCSASQG IRNYLNWYQQRPDGTVKLLIYYTSSLHS GVPSRFSGSGSGTDYSLTISNLEPEDIA TYYCQQYSKLPWTFGGGTKLEIK 14 VH MCSP EVQLQESGPGLVKPSQSLSLTCSVTGYS ITSGYYWNWIRQFPGNKLEWMGYITYDG SNNYNPSLKNRISITRDTSKNQFFLKLN SVTTEDTATYYCADFDYWGQGTTLTVSS 15 CL MCSP RTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC 16 CH1 MCSP ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCD 17 LIGHT CHAIN DIVLTQSPSSLSASLGDRVTISCSASQG MCSP IRNYLNWYQQRPDGTVKLLIYYTSSLHS GVPSRFSGSGSGTDYSLTISNLEPEDIA TYYCQQYSKLPWTFGGGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 18 HEAVY CHAIN EVQLQESGPGLVKPSQSLSLTCSVTGYS MCSP ITSGYYWNWIRQFPGNKLEWMGYITYDG SNNYNPSLKNRISITRDTSKNQFFLKLN SVTTEDTATYYCADFDYWGQGTTLTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCD 19 VL CD3_((V9)) DIQMT QSPSSLSASVGDRVTITCRASQD IRNYLNWYQQKPGKAPKLLIYYTSRLES GVPSRFSGSGSGTDYTLTISSLQPEDFA TYYCQQGNTLPWTFGQGTKVEIK 20 VH CD3_((V9)) EVQLVESGGGLVQPGGSLRLSCAASGYS FTGYTMNWVRQAPGKGLEWVALINPYKG VSTYNQKFKDRFTISVDKSKNTAYLQMN SLRAEDTAVYYCARSGYYGDSDWYFDVW GQGTLVTVSS 21 CL CD3_((V9)) VAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC 22 CH CD3_((V9)) ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSC 23 LIGHT CHAIN EVQLVESGGGLVQPGGSLRLSCAASGYS CD3_((V9)) (VHCL) FTGYTMNWVRQAPGKGLEWVALINPYKG VSTYNQKFKDRFTISVDKSKNTAYLQMN SLRAEDTAVYYCARSGYYGDSDWYFDVW GQGTLVTVSSASVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 24 HEAVY CHAIN DIQMTQSPSSLSASVGDRVTITCRASQD CD3_((V9)) (VLCH1) IRNYLNWYQQKPGKAPKLLIYYTSRLES GVPSRFSGSGSGTDYTLTISSLQPEDFA TYYCQQGNTLPWTFGQGTKVEIK SSAST KGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSC 35 VL CD3_((H2C)) QTVVTQEPSLTVSPGGTVTLTCGSSTGA VTSGYYPNWVQQKPGQAPRGLIGGTKFL APGTPARFSGSLLGGKAALTLSGVQPED EAEYYCALWYSNRWVFGGGTKLTVL 36 VH CD3_((H2C)) EVQLVESGGGLVQPGGSLKLSCAASGFT FNKYAMNWVRQAPGKGLEWVARIRSKYN NYATYYADSVKDRFTISRDDSKNTAYLQ MNNLKTEDTAVYYCVRHGNFGNSYISYW AYWGQGTLVTVSS 37 CL CD3_((H2C)) VAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC 38 CH1 CD3_((H2C)) ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSC 39 LIGHT CHAIN EVQLVESGGGLVQPGGSLKLSCAASGFT CD3_((H2C))(VHCL) FNKYAMNWVRQAPGKGLEWVARIRSKYN NYATYYADSVKDRFTISRDDSKNTAYLQ MNNLKTEDTAVYYCVRHGNFGNSYISYW AYWGQGTLVTVSSASVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC 40 HEAVY CHAIN QTVVTQEPSLTVSPGGTVTLTCGSSTGA CD3_((H2C))(VLCH1) VTSGYYPNWVQQKPGQAPRGLIGGTKFL APGTPARFSGSLLGGKAALTLSGVQPED EAEYYCALWYSNRWVFGGGTKLTVL SSA STKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSC 25 FAB (MCSP)- EVQLQESGPGLVKPSQSLSLTCSVTGYS XFAB (CD3_((V9))) ITSGYYWNWIRQFPGNKLEWMGYITYDG (VH-CH1-VL- SNNYNPSLKNRISITRDTSKNQFFLKLN CH1) SVTTEDTATYYCADFDYWGQGTTLTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDGGGGSGGG GS DIQMTQSPSSLSASVGDRVTITCRAS QDIRNYLNWYQQKPGKAPKLLIYYTSRL ESGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQGNTLPWTFGQGTKVEIK SSA STKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSC 26 FAB (MCSP)- EVQLQESGPGLVKPSQSLSLTCSVTGYS FAB (MCSP)- ITSGYYWNWIRQFPGNKLEWMGYITYDG XFAB (CD3_((V9))) SNNYNPSLKNRISITRDTSKNQFFLKLN (VH-CH1-VH SVTTEDTATYYCADFDYWGQGTTLTVSS CH1-VL-CH1) ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDGGGGSGGG GSEVQLQESGPGLVKPSQSLSLTCSVTG YSITSGYYWNWIRQFPGNKLEWMGYITY DGSNNYNPSLKNRISITRDTSKNQFFLK LNSVTTEDTATYYCADFDYWGQGTTLTV SSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDGGGGSG GGGS DIQMTQSPSSLSASVGDRVTITCR ASQDIRNYLNWYQQKPGKAPKLLIYYTS RLESGVPSRFSGSGSGTDYTLTISSLQP EDFATYYCQQGNTLPWTFGQGTKVEIK S SASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSC 27 FAB (MCSP)- EVQLQESGPGLVKPSQSLSLTCSVTGYS XFAB (CD3_((V9)))- ITSGYYWNWIRQFPGNKLEWMGYITYDG FAB (MCSP) SNNYNPSLKNRISITRDTSKNQFFLKLN (VH-CH1-VL- SVTTEDTATYYCADFDYWGQGTTLTVSS CH1-VH-CH1) ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDGGGGSGGG GS DIQMTQSPSSLSASVGDRVTITCRAS QDIRNYLNWYQQKPGKAPKLLIYYTSRL ESGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQGNTLPWTFGQGTKVEIK SSA STKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCGGGGSGGGGS EVQLQESGPGLVKPSQSLSLTCSVTGYS ITSGYYWNWIRQFPGNKLEWMGYITYDG SNNYNPSLKNRISITRDTSKNQFFLKLN SVTTEDTATYYCADFDYWGQGTTLTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSC 28 LINKER 1 GGGGSGGGGS 41 FAB (MCSP)- EVQLQESGPGLVKPSQSLSLTCSVTGYS FAB (MCSP)- ITSGYYWNWIRQFPGNKLEWMGYITYDG XFAB (CD3_((H2C))) SNNYNPSLKNRISITRDTSKNQFFLKLN (VH-CH1-VH- SVTTEDTATYYCADFDYWGQGTTLTVSS CH1-VL-CH1) ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDGGGGSGGG GSEVQLQESGPGLVKPSQSLSLTCSVTG YSITSGYYWNWIRQFPGNKLEWMGYITY DGSNNYNPSLKNRISITRDTSKNQFFLK LNSVTTEDTATYYCADFDYWGQGTTLTV SSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDGGGGSG GGGS QTVVTQEPSLTVSPGGTVTLTCGS STGAVTSGYYPNWVQQKPGQAPRGLIGG TKFLAPGTPARFSGSLLGGKAALTLSGV QPEDEAEYYCALWYSNRWVFGGGTKLTV L SSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSC 42 Murine LIGHT EVQLVESGGGLVQPGKSLKLSCEASGFT CHAIN CD3_((2C11)) FSGYGMHWVRQAPGRGLESVAYITSSSI (VHCL) NIKYADAVKGRFTVSRDNAKNLLFLQMN ILKSEDTAMYYCARFDWDKNYWGQGTMV TVSSASVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC 43 Murine DIQMTQSPSSLPASLGDRVTINCQASQD XFAB (CD3_((2C11)))- ISNYLNWYQQKPGKAPKLLIYYTNKLAD FAB (MCSP)- GVPSRFSGSGSGRDSSFTISSLESEDIG FAB (MCSP) SYYCQQYYNYPWTFGPGTKLEIKSSAST (VL-CH1-VH- KGPSVFPLAPSSKSTSGGTAALGCLVKD CH1-VH-CH1) YFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCGGGGSGGGGSEV QLQESGPGLVKPSQSLSLTCSVTGYSIT SGYYWNWIRQFPGNKLEWMGYITYDGSN NYNPSLKNRISITRDTSKNQFFLKLNSV TTEDTATYYCADFDYWGQGTTLTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDGGGGSGGGGS EVQLQESGPGLVKPSQSLSLTCSVTGYS ITSGYYWNWIRQFPGNKLEWMGYITYDG SNNYNPSLKNRISITRDTSKNQFFLKLN SVTTEDTATYYCADFDYWGQGTTLTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCD 68 GA201 CDR1 VH DYKIH 69 GA201 CDR2 VH YFNPNSGYSTYAQKFQG 70 GA201 CDR3 VH LSPGGYYVMDA 71 GA201 CDR1 VL RASQGINNYLN 72 GA201 CDR2 VL NTNNLQT 73 GA201 CDR3 VL LQHNSFPT 74 GA201 VH QVQLVQSGAEVKKPGSSVKVSCKASGFT FTDYKIHWVRQAPGQGLEWMGYFNPNSG YSTYAQKFQGRVTITADKSTSTAYMELS SLRSEDTAVYYCARLSPGGYYVMDAWGQ GTTVTVSS 75 GA201 VL DIQMTQSPSSLSASVGDRVTITCRASQG INNYLNWYQQKPGKAPKRLIYNTNNLQT GVPSRFSGSGSGTEFTLTISSLQPEDFA TYYCLQHNSFPTFGQGTKLEIK 76 3F2 CDR1 VH SYAMS 77 3F2 CDR2 VH AISGSGGSTYYADSVK 78 3F2 CDR3 VH YCAKGWFG 79 3F2 CDR1 VL RASQSVTSSYL 80 3F2 CDR2 VL NVGSRRA 81 3F2 CDR3 VL CQQGIMLPP 82 3F2 VH EVQLLESGGGLVQPGGSLRLSCAASGFT FSSYAMSWVRQAPGKGLEWVSAISGSGG STYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKGWFGGFNYWGQGTL VTVSS 83 3F2 VL EIVLTQSPGTLSLSPGERATLSCRASQS VTSSYLAWYQQKPGQAPRLLINVGSRRA TGIPDRFSGSGSGTDFTLTISRLEPEDF AVYYCQQGIMLPPTFGQGTKVEIK 84 CH1A1A CDR1 VH EFGMN 85 CH1A1A CDR2 VH WINTKTGEATYVEEFKG 86 CH1A1A CDR3 VH WDFAYYVEAMDY 87 CH1A1A CDR1 VL KASAAVGTYVA 88 CH1A1A CDR2 VL SASYRKR 89 CH1A1A CDR3 VL HQYYTYPLFT 90 CH1A1A VH QVQLVQSGAEVKKPGASVKVSCKASGYT FTEFGMNWVRQAPGQGLEWMGWINTKTG EATYVEEFKGRVTFTTDTSTSTAYMELR SLRSDDTAVYYCARWDFAYYVEAMDYWG QGTTVTVSS 91 CH1A1A VL DIQMTQSPSSLSASVGDRVTITCKASAA VGTYVAWYQQKPGKAPKLLIYSASYRKR GVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCHQYYTYPLFTFGQGTKLEIK 92 Anti-CD33 CDR1 VH GYTITDSNIH 93 Anti-CD33 CDR2 VH YIYPYNGGTDYNQ 94 Anti-CD33 CDR3 VH GNPWLAY 95 Anti-CD33 CDR1 VL RASESLDNYGIRFLT 96 Anti-CD33 CDR1 VL AASNQGS 97 Anti-CD33 CDR1 VL QQTKEVPWS 98 Anti-CD33 VH EVQLVQSGAEVKKPGSSVKVSCKASGYT ITDSNIHWVRQAPGQSLEWIGYIYPYNG GTDYNQKFKNRATLTVDNPTNTAYMELS SLRSEDTAFYYCVNGNPWLAYWGQGTLV TVSS 99 Anti-CD33 VL DIQLTQSPSTLSASVGDRVTITCRASES LDNYGIRFLTWFQQKPGKAPKLLMYAAS NQGSGVPSRFSGSGSGTEFTLTISSLQP DDFATYYCQQTKEVPWSFGQGTKVEVK 100 Light Chain DIQMTQSPSSLSASVGDRVTITCRASES antiCD33 VDNYGISFMNWFQQKPGKAPKLLIYAAS NQGSGVPSRFSGSGSGTDFTLTISSLQP DDFATYYCQQSKEVPWTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 101 Light Chain EVQLVESGGGLVQPGGSLRLSCAASGYS CD3_((V9)) FTGYTMNWVRQAPGKGLEWVALINPYKG (VH-CL) VSTYNQKFKDRFTISVDKSKNTAYLQMN SLRAEDTAVYYCARSGYYGDSDWYFDVW GQGTLVTVSSASVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 102 Fab (CD33)- QVQLVQSGAEVKKPGSSVKVSCKASGYT XFab (CD3_((V9))) FTDYNMHWVRQAPGQGLEWIGYIYPYNG (VH-CH1-VL- GTGYNQKFKSKATITADESTNTAYMELS CH1) SLRSEDTAVYYCARGRPAMDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDGGGG SGGGGSDIQMTQSPSSLSASVGDRVTIT CRASQDIRNYLNWYQQKPGKAPKLLIYY TSRLESGVPSRFSGSGSGTDYTLTISSL QPEDFATYYCQQGNTLPWTFGQGTKVEI KSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTF PAVL 145 Linker 2 EPKSCGGGGSGGGGS 146 Linker 3 EPKSCDGGGGSGGGGS 147 Linker 4 GGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGG 148 Linker 5 SGGGSGGGSEGGGSEGGGSEGGGSEGGG SGGGSG 149 (scFv)2 DIVLTQSPSSLSASLGDRVTISCSASQG antiMCSP/anti IRNYLNWYQQRPDGTVKLLIYYTSSLHS huCD3e GVPSRFSGSGSGTDYSLTISNLEPEDIA (MCSP(VL- VH)- TYYCQQYSKLPWTFGGGTKLEIKGGGGS CD3_((V9))(VH-VL)) GGGGSGGGGSEVQLQESGPGLVKPSQSL SLTCSVTGYSITSGYYWNWIRQFPGNKL EWMGYITYDGSNNYNPSLKNRISITRDT SKNQFFLKLNSVTTEDTATYYCADFDYW GQGTTLTVSSGGGGSEVQLVESGGGLVQ PGGSLRLSCAASGYSFTGYTMNWVRQAP GKGLEWVALINPYKGVSTYNQKFKDRFT ISVDKSKNTAYLQMNSLRAEDTAVYYCA RSGYYGDSDWYFDVWGQGTLVTVSSVEG GSGGSGGSGGSGGVDDIQMTQSPSSLSA SVGDRVTITCRASQDIRNYLNWYQQKPG KAPKLLIYYTSRLESGVPSRFSGSGSGT DYTLTISSLQPEDFATYYCQQGNTLPWT FGQGTKVEIKHHHHHH 151 Light Chain DIQLTQSPSTLSASVGDRVTITCRASES antiCD33_((Myelotarg)) LDNYGIRFLTWFQQKPGKAPKLLMYAAS NQGSGVPSRFSGSGSGTEFTLTISSLQP DDFATYYCQQTKEVPWSFGQGTKVEVKR TVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 152 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQD CD3_((V9)) IRNYLNWYQQKPGKAPKLLIYYTSRLES (VL-CH1) GVPSRFSGSGSGTDYTLTISSLQPEDFA TYYCQQGNTLPWTFGQGTKVEIKSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSC 153 Fab EVQLVQSGAEVKKPGSSVKVSCKASGYT (CD33(Myelotarg)- ITDSNIHWVRQAPGQSLEWIGYIYPYNG XFab (CD3_((V9))) GTDYNQKFKNRATLTVDNPTNTAYMELS (VH-CH1-VH- SLRSEDTAFYYCVNGNPWLAYWGQGTLV CL) TVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDGGGG SGGGGSEVQLVESGGGLVQPGGSLRLSC AASGYSFTGYTMNWVRQAPGKGLEWVAL INPYKGVSTYNQKFKDRFTISVDKSKNT AYLQMNSLRAEDTAVYYCARSGYYGDSD WYFDVWGQGTLVTVSSASVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC 157 CD3_((CH2527)) VL QAVVTQESALTTSPGETVTLTCRSSTGA VTTSNYANWVQEKPDHLFTGLIGGTNKR APGVPARFSGSLIGDKAALTITGAQTED EAIYFCALWYSNLWVFGGGTKLTVL 158 CD3_((CH2527)) VH EVQLVESGGGLVQPKGSLKLSCAASGFT FNTYAMNWVRQAPGKGLEWVARIRSKYN NYATYYADSVKDRFTISRDDSQSILYLQ MNNLKTEDTAMYYCVRHGNFGNSYVSWF AYWGQGTLVTVSA 159 CEA_((CH1A1A(98/99))) QVQLVQSGAEVKKPGASVKVSCKASGYT VH FTEFGMNWVRQAPGQGLEWMGWINTKTG EATYVEEFKGRVTFTTDTSTSTAYMELR SLRSDDTAVYYCARWDFAYYVEAMDYWG QGTTVTVSS 160 CEA_((CH1A1A(98/99))) DIQMTQSPSSLSASVGDRVTITCKASAA VL VGTYVAWYQQKPGKAPKLLIYSASYRKR GVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCHQYYTYPLFTFGQGTKLEIK 161 MCSP_((M4-3 ML2)) VHQVQLQESGPGLVKPSQTLSLTCTVSG GSITSGYYWNWIRQHPGKGLEWIGYITY DGSNNYNPSLKSRVTISRDTSKNQFSLK LSSVTAADTAVYYCADFDYWGQGTLVTV SS 162 MCSP_((M4-3 ML2)) VLDIQMTQSPSSLSASVGDRVTITCRAS QGIRNYLNWYQQKPGKAPKLLIYYTSSL HSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQYSKLPWTFGQGTKVEIK 163 VLCL(MCSP) - EVQLVESGGGLVQPGGSLRLSCAASGYS (G4S)2- FTGYTMNWVRQAPGKGLEWVALINPYKG VHCL(CD3_(V9)) VSTYNQKFKDRFTISVDKSKNTAYLQMN SLRAEDTAVYYCARSGYYGDSDWYFDVW GQGTLVTVSSASVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSF NRGECDGGGGSGGGGSDIVLTQSPSSLS ASLGDRVTISCSASQGIRNYLNWYQQRP DGTVKLLIYYTSSLHSGVPSRFSGSGSG TDYSLTISNLEPEDIATYYCQQYSKLPW TFGGGTKLEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 164 VHCH1(MCSP)- EVQLQESGPGLVKPSQSLSLTCSVTGYS (G4S)2- ITSGYYWNWIRQFPGNKLEWMGYITYDG VLCH1(CD3_(V9)) SNNYNPSLKNRISITRDTSKNQFFLKLN SVTTEDTATYYCADFDYWGQGTTLTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDGGGGSGGG GSDIQMTQSPSSLSASVGDRVTITCRAS QDIRNYLNWYQQKPGKAPKLLIYYTSRL ESGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQGNTLPWTFGQGTKVEIKSSA STKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSC Protein Sequences: Humanised CD3 Binders and Affinity Matured MCSP Binders

CD3 _(CH2527) SEQ ID NO. Heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAP 167 “CD3 _(CH2527 (VH)_3-23(12))” GKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK Light chain QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK 168 “CD3 _(CH2527 (VL)_7-46(13))” PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP EDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPP SSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET TTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTV EKTVAPTECS VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAP 169 “CD3 _(CH2527 (VH)_3-23(12))” GKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTV SS VH CDR H1 TYAMN 170 “CD3 _(CH2527 (VH)_3-23(12))” VH CDR H2 RIRSKYNNYATYYADSVKG 171 “CD3 _(CH2527 (VH)_3-23(12))” VH CDR H3 HGNFGNSYVSWFAY 172 “CD3 _(CH2527 (VH)_3-23(12))” VL QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK 173 “CD3 _(CH2527 (VL)_7-46(13))” PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP EDEAEYYCALWYSNLWVFGGGTKLTVL VL CDR L1 GSSTGAVTTSNYAN 174 “CD3 _(CH2527 (VL)_7-46(13))” VL CDR L2 GTNKRAP 175 “CD3 _(CH2527 (VL)_7-46(13))” VL CDR L3 ALWYSNLWV 176 “CD3 _(CH2527 (VL)_7-46(13))” MCSP _(M4-3(C1)ML2(G3)) SEQ ID NO Heavy chain QVQLQESGPGLVKPSQTLSLTCTVSGGSITSGYYWNWIRQH 177 ,,MCSP _(M4-3(C1))” PGKGLEWIGYITFDGSNNYNPSLKSRVTISRDTSKNQFSLK LSSVTAADTAVYYCADFDYWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light chain DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLNWYQQKPG 178 ,,MCSP _(ML2(G3))” KAPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQYSALPWTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC VH QVQLQESGPGLVKPSQTLSLTCTVSGGSITSGYYWNWIRQH 179 ,,MCSP _(M4-3(C1))” PGKGLEWIGYITFDGSNNYNPSLKSRVTISRDTSKNQFSLK LSSVTAADTAVYYCADFDYWGQGTLVTVSS VH CDR H1 SGYYWN 180 ,,MCSP _(M4-3(C1))” VH CDR H2 YITFDGSNNYNPSLKS 181 ,,MCSP _(M4-3(C1))” VH CDR H3 FDY 6 ,,MCSP _(M4-3(C1))” VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLNWYQQKPG 182 ,,MCSP _(ML2(G3))” KAPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPED FATYYCQQYSALPWTFGQGTKVEIK VL CDR L1 RASQGIRNYLN 183 ,,MCSP _(ML2(G3))” VL CDR L2 YTSSLHS 2 ,,MCSP _(ML2(G3))” VL CDR L3 QQYSALPWT 184 ,,MCSP _(ML2(G3))” CD3 _(VL)_7-43(11)/VH_3-23(12) SEQ ID NO. VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAP 169 “CD3 _(CH2527(VH)_3-23(12))” GKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTV SS VH CDR H1 TYAMN 170 “CD3 _(CH2527(VH)_3-23(12))” VH CDR H2 RIRSKYNNYATYYADSVKG 171 “CD3 _(CH2527(VH)_3-23(12))” VH CDR H3 HGNFGNSYVSWFAY 172 “CD3 _(CH2527(VH)_3-23(12))” VL QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQK 185 “CD3 _(CH2527(VL)_7-43(11))” PGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQP EDEAEYYCALWYSNLWVFGGGTKLTVLSS VL CDR L1 GSSTGAVTTSNYAN 174 “CD3 _(CH2527(VL)_7-43(11))” VL CDR L2 GTNKRAP 175 “CD3 _(CH2527(VL)_7-43(11))” VL CDR L3 ALWYSNLWV 176 “CD3 _(CH2527(VL)_7-43(11))” CD3 _(VL)_7-43(11)/VHcomboA49SV93A VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAP 186 ,,CD3 _(CH2527(VHcomboA49SV93A))” GKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTV SS VH CDR H1 TYAMN 170 ,,CD3 _(CH2527(VHcomboA49SV93A))” VH CDR H2 RIRSKYNNYATYYADSVKG 171 ,,CD3 _(CH2527(VHcomboA49SV93A))” VH CDR H3 HGNFGNSYVSWFAY 172 ,,CD3 _(CH2527(VHcomboA49SV93A))” VL QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQK 185 “CD3 _(CH2527(VL)_7-43(11))” PGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQP EDEAEYYCALWYSNLWVFGGGTKLTVLSS VL CDR L1 GSSTGAVTTSNYAN 174 “CD3 _(CH2527(VL)_7-43(11))” VL CDR L2 GTNKRAP 175 “CD3 _(CH2527(VL)_7-43(11))” VL CDR L3 ALWYSNLWV 176 “CD3 _(CH2527(VL)_7-43(11))” CD3 _(VL)_7-46(13)/VHcomboA49SV93A VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAP 186 ,,CD3 _(CH2527(VHcomboA49SV93A))” GKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTV SS VH CDR H1 TYAMN 170 ,,CD3 _(CH2527(VHcomboA49SV93A))” VH CDR H2 RIRSKYNNYATYYADSVKG 171 ,,CD3 _(CH2527(VHcomboA49SV93A))” VH CDR H3 HGNFGNSYVSWFAY 172 ,,CD3 _(CH2527(VHcomboA49SV93A))” VL QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK 173 “CD3 _(CH2527(VL)_7-46(13))” PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP EDEAEYYCALWYSNLWVFGGGTKLTVL VL CDR L1 GSSTGAVTTSNYAN 174 “CD3 _(CH2527(VL)_7-46(13))” VL CDR L2 GTNKRAP 175 “CD3 _(CH2527(VL)_7-46(13))” VL CDR L3 ALWYSNLWV 176 “CD3 _(CH2527(VL)_7-46(13))” CD3 _(VL)_7-43(11)/VHcomboA49SV93AR94K VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAP 187 ,,CD3 _(CH2527) GKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLY _((VHcomboA49SV93AR94K))” LQMNSLRAEDTAVYYCAKHGNFGNSYVSWFAYWGQGTLVTV SS VH CDR H1 TYAMN 170 ,,CD3 _(CH2527) _((VHcomboA49SV93AR94K))” VH CDR H2 RIRSKYNNYATYYADSVKG 171 ,,CD3 _(CH2527) _((VHcomboA49SV93AR94K))” VH CDR H3 HGNFGNSYVSWFAY 172 ,,CD3 _(CH2527) _((VHcomboA49SV93AR94K))” VL QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQK 185 “CD3 _(CH2527(VL)_7-43(11))” PGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQP EDEAEYYCALWYSNLWVFGGGTKLTVLSS VL CDR L1 GSSTGAVTTSNYAN 174 “CD3 _(CH2527(VL)_7-43(11))” VL CDR L2 GTNKRAP 175 “CD3 _(CH2527(VL)_7-43(11))” VL CDR L3 ALWYSNLWV 176 “CD3 _(CH2527(VL)_7-43(11))” CD3 _(VL)_7-46(13)/VHcomboA95SV93AR94K VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAP 187 ,,CD3 _(CH2527) GKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLY _((VHcomboA49SV93AR94K))” LQMNSLRAEDTAVYYCAKHGNFGNSYVSWFAYWGQGTLVTV SS VH CDR H1 TYAMN 170 ,,CD3 _(CH2527) _((VHcomboA49SV93AR94K))” VH CDR H2 RIRSKYNNYATYYADSVKG 171 ,,CD3 _(CH2527) _((VHcomboA49SV93AR94K))” VH CDR H3 HGNFGNSYVSWFAY 172 ,,CD3 _(CH2527) _((VHcomboA49SV93AR94K))” VL QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK 173 “CD3 _(CH2527(VL)_7-46(13))” PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP EDEAEYYCALWYSNLWVFGGGTKLTVL VL CDR L1 GSSTGAVTTSNYAN 174 “CD3 _(CH2527(VL)_7-46(13))” VL CDR L2 GTNKRAP 175 “CD3 _(CH2527(VL)_7-46(13))” VL CDR L3 ALWYSNLWV 176 “CD3 _(CH2527(VL)_7-46(13))” MCSP SEQ ID NO VH QVQLQESGPGLVKPSQTLSLTCTVSGGSITSGYYWNWIRQH 188 ,,MCSP_( M4-3(D6))” PGKGLEWIGYITFDGKNNYNPSLKSRVTISRDTSKNQFSLK LSSVTAADTAVYYCADFDYWGQGTLVTVSS VH CDR H1 SGYYWN 180 ,,MCSP _(M4-3(D6))” VH CDR H2 ITFDGKNNYNPSLKS 189 ,,MCSP _(M4-3(D6))” VH CDR H3 FDY 6 ,,MCSP _(M4-3(D6))” VH QVQLQESGPGLVKPSQTLSLTCTVSGGSITDGYYWNWIRQH 190 ,,MCSP _(M4-3(A7))” PGKGLEWIGYITFDGRNNYNPSLKSRVTISRDTSKNQFSLK LSSVTAADTAVYYCADFDYWGQGTLVTVSS VH CDR H1 DGYYWN 191 ,,MCSP _(M4-3(A7))” VH CDR H2 ITFDGRNNYNPSLKS 192 ,,MCSP _(M4-3(A7))” VH CDR H3 FDY 6 ,,MCSP _(M4-3(A7))” VH QVQLQESGPGLVKPSQTLSLTCTVSGGSITSGYYWNWIRQH 193 ,,MCSP _(M4-3(B7))” PGKGLEWIGYITFDGINNYNPSLKSRVTISRDTSKNQFSLK LSSVTAADTAVYYCADFDYWGQGTLVTVSS VH CDR H1 SGYYWN 180 ,,MCSP _(M4-3(B7))” VH CDR H2 ITFDGINNYNPSLKS 194 ,,“MCSP _(M4-3(B7))” VH CDR H3 FDY 6 ,,MCSP _(M4-3(B7))” VH QVQLQESGPGLVKPSQTLSLTCTVSGGSITSGYYWNWIRQH 195 ,,MCSP _(M4-3(B8))” PGKGLEWIGYITFDGRNNYNPSLKSRVTISRDTSKNQFSLK LSSVTAADTAVYYCADFDYWGQGTLVTVSS VH CDR H1 SGYYWN 180 ,,MCSP _(M4-3(B8))” VH CDR H2 ITFDGRNNYNPSLKS 192 ,,MCSP _(M4-3(B8))” VH CDR H3 FDY 6 ,,MCSP _(M4-3(B8))” Parental VH MCSP _(M4-3) QVQLQESGPGLVKPSQTLSLTCTVSGGSITSGYYWNWIRQH 161 PGKGLEWIGYITYDGSNNYNPSLKSRVTISRDTSKNQFSLK LSSVTAADTAVYYCADFDYWGQGTLVTVSS VL DIQMTQSPSSLSASVGDRVTITCRASYGIRGYLNWYQQKPG 196 ,,MCSP _(ML2(E10))” KAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPED FATYHCQQYSKLPWTFGQGTKVEIK VL CDR L1 RASYGIRGYLN 197 ,,MCSP _(ML2(E10))” VL CDR L2 YTSSLHS 2 ,,MCSP _(ML2(E10))” VL CDR L3 QQYSKLPWT 3 ,,MCSP _(ML2(E10))” VL DIQMTQSPSSLSASVGDRVTITCRASYGIRGYLNWYQQKPG 198 ,,MCSP _(ML2(E10-G3))” KAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPED FATYHCQQYSALPWTFGQGTKVEIK VL CDR L1 RASYGIRGYLN 197 ,,MCSP _(ML2(E10-G3))” VL CDR L2 YTSSLHS 2 ,,MCSP _(ML2(E10-G3))” VL CDR L3 QQYSKLPWT 3 ,,MCSP _(ML2(E10-G3))” VL DIQMTQSPSSLSASVGDRVTITCRASRGIREYLNWYQQKPG 199 ,,MCSP _(ML2(C5))” KAPKLLIYYTGSLHSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQYSELPWTFGQGTKVEIK VL CDR L1 RASRGIREYLN 200 ,,MCSP _(ML2(C5))” VL CDR L2 YTGSLHS 201 ,,MCSP _(ML2(C5))” VL CDR L3 QQYSELPWT 202 ,,MCSP _(ML2(C5))” VL DIQMTQSPSSLSASVGDRVTITCRASRGIREYLNWYQQKPG 203 ,,MCSP _(ML2(C5-G3))” KAPKLLIYYTGSLHSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQYSALPWTFGQGTKVEIK VL CDR Ll RASRGIREYLN 200 ,,MCSP _(ML2(C5-G3))” VL CDR L2 YTGSLHS 201 ,,MCSP _(ML2(C5-G3))” VL CDR L3 QQYSKLPWT 3 ,,MCSP _(ML2(C5-G3))” Parental VL MCSP _(ML2) DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLNWYQQKPG 162 KAPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQYSKLPWTFGQGTKVEIK

DNA Sequences

SEQ ID. NO. Description Sequence 44 VL MCSP GATATTGTGCTCACACAGTCTCCATCCTCCC TGTCTGCCTCTCTGGGAGACAGAGTCACCAT CAGTTGCAGTGCAAGTCAGGGCATTAGAAAT TATTTAAACTGGTATCAGCAGAGACCAGATG GAACTGTTAAACTCCTGATCTATTACACATC AAGTTTACACTCAGGAGTCCCATCAAGGTTC AGTGGCAGTGGGTCTGGGACAGATTATTCTC TCACCATCAGCAACCTGGAACCTGAAGATAT TGCCACTTACTATTGTCAGCAGTATAGTAAG CTTCCTTGGACGTTCGGTGGAGGCACCAAGC TGGAAATCAAA 45 VH MCSP GAGGTGCAGCTGCAGGAATCTGGCCCTGGCC TGGTCAAGCCAAGCCAGAGTCTGAGCCTGAC CTGCAGCGTGACCGGCTACAGCATTACCAGC GGCTACTACTGGAACTGGATTCGGCAGTTCC CCGGCAATAAGCTGGAATGGATGGGCTACAT CACCTACGACGGCAGCAACAACTACAACCCC AGCCTGAAGAACCGGATCAGCATCACCCGGG ACACCAGCAAGAACCAGTTCTTCCTGAAGCT GAACAGCGTGACCACCGAGGACACCGCCACA TACTATTGCGCCGACTTCGACTACTGGGGCC AGGGCACCACCCTGACCGTGTCCAGC 46 CL MCSP CGTACGGTGGCTGCACCATCTGTCTTCATCT TCCCGCCATCTGATGAGCAGTTGAAATCTGG AACTGCCTCTGTTGTGTGCCTGCTGAATAAC TTCTATCCCAGAGAGGCCAAAGTACAGTGGA AGGTGGATAACGCCCTCCAATCGGGTAACTC CCAGGAGAGTGTCACAGAGCAGGACAGCAAG GACAGCACCTACAGCCTCAGCAGCACCCTGA CGCTGAGCAAAGCAGACTACGAGAAACACAA AGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAAGAGCTTCAACA GGGGAGAGTGTTAG 47 CH1 MCSP GCCAGCACAAAGGGCCCTAGCGTGTTCCCTC TGGCCCCCAGCAGCAAGAGCACAAGCGGCGG AACAGCCGCCCTGGGCTGCCTCGTGAAGGAC TACTTCCCCGAGCCCGTGACAGTGTCTTGGA ACAGCGGAGCCCTGACAAGCGGCGTGCACAC CTTCCCTGCCGTGCTGCAGAGCAGCGGCCTG TACTCCCTGAGCAGCGTGGTCACCGTGCCTA GCAGCAGCCTGGGCACCCAGACCTACATCTG CAACGTGAACCACAAGCCCAGCAACACCAAA GTGGACAAGAAGGTGGAGCCCAAGAGCTGTG AT 48 LIGHT CHAIN ATGGACATGAGGGTCCCCGCTCAGCTCCTGG MCSP GCCTCCTGCTGCTCTGGTTCCCAGGTGCCAG GTGTGATATTGTGCTCACACAGTCTCCATCC TCCCTGTCTGCCTCTCTGGGAGACAGAGTCA CCATCAGTTGCAGTGCAAGTCAGGGCATTAG AAATTATTTAAACTGGTATCAGCAGAGACCA GATGGAACTGTTAAACTCCTGATCTATTACA CATCAAGTTTACACTCAGGAGTCCCATCAAG GTTCAGTGGCAGTGGGTCTGGGACAGATTAT TCTCTCACCATCAGCAACCTGGAACCTGAAG ATATTGCCACTTACTATTGTCAGCAGTATAG TAAGCTTCCTTGGACGTTCGGTGGAGGCACC AAGCTGGAAATCAAACGTACGGTGGCTGCAC CATCTGTCTTCATCTTCCCGCCATCTGATGA GCAGTTGAAATCTGGAACTGCCTCTGTTGTG TGCCTGCTGAATAACTTCTATCCCAGAGAGG CCAAAGTACAGTGGAAGGTGGATAACGCCCT CCAATCGGGTAACTCCCAGGAGAGTGTCACA GAGCAGGACAGCAAGGACAGCACCTACAGCC TCAGCAGCACCCTGACGCTGAGCAAAGCAGA CTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCA CAAAGAGCTTCAACAGGGGAGAGTGTTAG 49 HEAVY ATGGGATGGAGCTGTATCATCCTCTTCTTGG CHAIN MCSP TAGCAACAGCTACCGGTGTGCATTCGGAGGT GCAGCTGCAGGAATCTGGCCCTGGCCTGGTC AAGCCAAGCCAGAGTCTGAGCCTGACCTGCA GCGTGACCGGCTACAGCATTACCAGCGGCTA CTACTGGAACTGGATTCGGCAGTTCCCCGGC AATAAGCTGGAATGGATGGGCTACATCACCT ACGACGGCAGCAACAACTACAACCCCAGCCT GAAGAACCGGATCAGCATCACCCGGGACACC AGCAAGAACCAGTTCTTCCTGAAGCTGAACA GCGTGACCACCGAGGACACCGCCACATACTA TTGCGCCGACTTCGACTACTGGGGCCAGGGC ACCACCCTGACCGTGTCCAGCGCCAGCACAA AGGGCCCTAGCGTGTTCCCTCTGGCCCCCAG CAGCAAGAGCACAAGCGGCGGAACAGCCGCC CTGGGCTGCCTCGTGAAGGACTACTTCCCCG AGCCCGTGACAGTGTCTTGGAACAGCGGAGC CCTGACAAGCGGCGTGCACACCTTCCCTGCC GTGCTGCAGAGCAGCGGCCTGTACTCCCTGA GCAGCGTGGTCACCGTGCCTAGCAGCAGCCT GGGCACCCAGACCTACATCTGCAACGTGAAC CACAAGCCCAGCAACACCAAAGTGGACAAGA AGGTGGAGCCCAAGAGCTGTGAT 50 VL CD3 _((V9)) GACATCCAGATGACCCAGAGCCCCTCTAGCC TGAGCGCCAGCGTGGGCGACAGAGTGACCAT CACCTGTCGGGCCAGCCAGGACATCAGAAAC TACCTGAACTGGTATCAGCAGAAGCCCGGCA AGGCCCCCAAGCTGCTGATCTACTACACCTC TAGACTGGAAAGCGGCGTGCCCAGCCGGTTT AGCGGCAGCGGCTCCGGCACCGACTACACCC TGACCATCAGCAGCCTGCAGCCCGAGGACTT CGCCACCTACTACTGCCAGCAGGGCAACACA CTCCCCTGGACCTTCGGCCAGGGCACCAAGG TGGAGATCAAGTCCAGC 51 VH CD3 _((V9)) GAGGTGCAGCTGGTCGAGAGCGGAGGCGGCC TGGTGCAGCCTGGCGGCAGCCTGAGACTGAG CTGCGCCGCCAGCGGCTACAGCTTCACCGGC TACACCATGAACTGGGTCCGGCAGGCACCTG GCAAGGGACTGGAATGGGTGGCCCTGATCAA CCCCTACAAGGGCGTGAGCACCTACAACCAG AAGTTCAAGGACCGGTTCACCATCAGCGTGG ACAAGAGCAAGAACACCGCCTATCTGCAGAT GAACAGCCTGCGGGCCGAGGACACCGCCGTG TACTACTGCGCCAGAAGCGGCTACTACGGCG ACAGCGACTGGTACTTCGACGTGTGGGGCCA GGGCACCCTCGTGACCGTGTCTAGC 52 CL CD3 _((V9)) GTGGCTGCACCATCTGTCTTCATCTTCCCGC CATCTGATGAGCAGTTGAAATCTGGAACTGC CTCTGTTGTGTGCCTGCTGAATAACTTCTAT CCCAGAGAGGCCAAAGTACAGTGGAAGGTGG ATAACGCCCTCCAATCGGGTAACTCCCAGGA GAGTGTCACAGAGCAGGACAGCAAGGACAGC ACCTACAGCCTCAGCAGCACCCTGACGCTGA GCAAAGCAGACTACGAGAAACACAAAGTCTA CGCCTGCGAAGTCACCCATCAGGGCCTGAGC TCGCCCGTCACAAAGAGCTTCAACAGGGGAG AGTGTTGA 53 CH CD3 _((V9)) ACCAAGGGCCCCTCCGTGTTCCCCCTGGCCC CCAGCAGCAAGAGCACCAGCGGCGGCACAGC CGCCCTCGGCTGCCTGGTCAAGGACTACTTC CCCGAGCCCGTGACCGTGTCCTGGAACAGCG GAGCCCTGACCTCCGGCGTGCACACCTTCCC CGCCGTGCTGCAGAGCAGCGGCCTGTACAGC CTGTCCAGCGTGGTCACCGTGCCCTCCAGCA GCCTGGGCACCCAGACCTACATCTGCAACGT GAACCACAAGCCCAGCAATACCAAGGTGGAC AAGAAGGTGGAGCCCAAGAGCTGCTGA 54 LIGHT CHAIN ATGGGATGGAGCTGTATCATCCTCTTCTTGG CD3_((V9)) TAGCAACAGCTACCGGTGTGCATTCCGAGGT (VHCL) GCAGCTGGTCGAGAGCGGAGGCGGCCTGGTG CAGCCTGGCGGCAGCCTGAGACTGAGCTGCG CCGCCAGCGGCTACAGCTTCACCGGCTACAC CATGAACTGGGTCCGGCAGGCACCTGGCAAG GGACTGGAATGGGTGGCCCTGATCAACCCCT ACAAGGGCGTGAGCACCTACAACCAGAAGTT CAAGGACCGGTTCACCATCAGCGTGGACAAG AGCAAGAACACCGCCTATCTGCAGATGAACA GCCTGCGGGCCGAGGACACCGCCGTGTACTA CTGCGCCAGAAGCGGCTACTACGGCGACAGC GACTGGTACTTCGACGTGTGGGGCCAGGGCA CCCTCGTGACCGTGTCTAGC GCTAGC GTGGC TGCACCATCTGTCTTCATCTTCCCGCCATCT GATGAGCAGTTGAAATCTGGAACTGCCTCTG TTGTGTGCCTGCTGAATAACTTCTATCCCAG AGAGGCCAAAGTACAGTGGAAGGTGGATAAC GCCCTCCAATCGGGTAACTCCCAGGAGAGTG TCACAGAGCAGGACAGCAAGGACAGCACCTA CAGCCTCAGCAGCACCCTGACGCTGAGCAAA GCAGACTACGAGAAACACAAAGTCTACGCCT GCGAAGTCACCCATCAGGGCCTGAGCTCGCC CGTCACAAAGAGCTTCAACAGGGGAGAGTGT TGA 55 HEAVY GACATCCAGATGACCCAGAGCCCCTCTAGCC CHAIN CD3_((V9)) TGAGCGCCAGCGTGGGCGACAGAGTGACCAT (VLCH1) CACCTGTCGGGCCAGCCAGGACATCAGAAAC TACCTGAACTGGTATCAGCAGAAGCCCGGCA AGGCCCCCAAGCTGCTGATCTACTACACCTC TAGACTGGAAAGCGGCGTGCCCAGCCGGTTT AGCGGCAGCGGCTCCGGCACCGACTACACCC TGACCATCAGCAGCCTGCAGCCCGAGGACTT CGCCACCTACTACTGCCAGCAGGGCAACACA CTCCCCTGGACCTTCGGCCAGGGCACCAAGG TGGAGATCAAGTCCAGC GCTAGC ACCAAGGG CCCCTCCGTGTTCCCCCTGGCCCCCAGCAGC AAGAGCACCAGCGGCGGCACAGCCGCCCTCG GCTGCCTGGTCAAGGACTACTTCCCCGAGCC CGTGACCGTGTCCTGGAACAGCGGAGCCCTG ACCTCCGGCGTGCACACCTTCCCCGCCGTGC TGCAGAGCAGCGGCCTGTACAGCCTGTCCAG CGTGGTCACCGTGCCCTCCAGCAGCCTGGGC ACCCAGACCTACATCTGCAACGTGAACCACA AGCCCAGCAATACCAAGGTGGACAAGAAGGT GGAGCCCAAGAGCTGCTGA 56 VL CD3 _((H2C)) CAGACCGTGGTGACACAGGAACCCAGCCTGA CCGTCTCCCCTGGCGGCACCGTGACCCTGAC CTGTGGAAGCAGCACAGGCGCCGTGACCAGC GGCTACTACCCCAACTGGGTGCAGCAGAAGC CCGGCCAGGCCCCTAGAGGACTGATCGGCGG CACCAAGTTTCTGGCCCCTGGCACCCCCGCC AGATTCTCTGGCTCTCTGCTGGGCGGCAAGG CCGCCCTGACACTGTCTGGCGTGCAGCCTGA GGACGAGGCCGAGTACTACTGCGCCCTGTGG TACAGCAACAGATGGGTGTTCGGCGGAGGCA CCAAGCTGACCGTGCTGAGCAGC 57 VH CD3 _((H2C)) GAGGTGCAGCTGGTGGAAAGCGGCGGAGGAC TGGTGCAGCCTGGCGGAAGCCTGAAGCTGTC TTGCGCCGCCAGCGGCTTCACCTTCAACAAA TACGCCATGAACTGGGTGCGCCAGGCCCCTG GCAAGGGACTGGAATGGGTGGCCCGGATCAG AAGCAAGTACAACAACTACGCCACCTACTAC GCCGACAGCGTGAAGGACCGGTTCACCATCA GCCGGGACGACAGCAAGAACACCGCCTACCT GCAGATGAACAACCTGAAAACCGAGGACACC GCCGTGTACTACTGCGTGCGGCACGGCAACT TCGGCAACAGCTACATCAGCTACTGGGCCTA CTGGGGACAGGGCACCCTGGTGACAGTGTCC AGC 58 CL CD3 _((H2C)) GTGGCTGCACCATCTGTCTTCATCTTCCCGC CATCTGATGAGCAGTTGAAATCTGGAACTGC CTCTGTTGTGTGCCTGCTGAATAACTTCTAT CCCAGAGAGGCCAAAGTACAGTGGAAGGTGG ATAACGCCCTCCAATCGGGTAACTCCCAGGA GAGTGTCACAGAGCAGGACAGCAAGGACAGC ACCTACAGCCTCAGCAGCACCCTGACGCTGA GCAAAGCAGACTACGAGAAACACAAAGTCTA CGCCTGCGAAGTCACCCATCAGGGCCTGAGC TCGCCCGTCACAAAGAGCTTCAACAGGGGAG AGTGTTGA 59 CH1 CD3 _((H2C)) ACCAAGGGCCCCTCCGTGTTCCCCCTGGCCC CCAGCAGCAAGAGCACCAGCGGCGGCACAGC CGCCCTCGGCTGCCTGGTCAAGGACTACTTC CCCGAGCCCGTGACCGTGTCCTGGAACAGCG GAGCCCTGACCTCCGGCGTGCACACCTTCCC CGCCGTGCTGCAGAGCAGCGGCCTGTACAGC CTGTCCAGCGTGGTCACCGTGCCCTCCAGCA GCCTGGGCACCCAGACCTACATCTGCAACGT GAACCACAAGCCCAGCAATACCAAGGTGGAC AAGAAGGTGGAGCCCAAGAGCTGCTGA 60 LIGHT CHAIN ATGGGATGGAGCTGTATCATCCTCTTCTTGG CD3 _((H2C)) TAGCAACAGCTACCGGTGTGCATTCCGAGGT (VHCL) GCAGCTGGTGGAAAGCGGCGGAGGACTGGTG CAGCCTGGCGGAAGCCTGAAGCTGTCTTGCG CCGCCAGCGGCTTCACCTTCAACAAATACGC CATGAACTGGGTGCGCCAGGCCCCTGGCAAG GGACTGGAATGGGTGGCCCGGATCAGAAGCA AGTACAACAACTACGCCACCTACTACGCCGA CAGCGTGAAGGACCGGTTCACCATCAGCCGG GACGACAGCAAGAACACCGCCTACCTGCAGA TGAACAACCTGAAAACCGAGGACACCGCCGT GTACTACTGCGTGCGGCACGGCAACTTCGGC AACAGCTACATCAGCTACTGGGCCTACTGGG GACAGGGCACCCTGGTGACAGTGTCCAGC GC TAGC GTGGCTGCACCATCTGTCTTCATCTT CCCGCCATCTGATGAGCAGTTGAAATCTGGA ACTGCCTCTGTTGTGTGCCTGCTGAATAACT TCTATCCCAGAGAGGCCAAAGTACAGTGGAA GGTGGATAACGCCCTCCAATCGGGTAACTCC CAGGAGAGTGTCACAGAGCAGGACAGCAAGG ACAGCACCTACAGCCTCAGCAGCACCCTGAC GCTGAGCAAAGCAGACTACGAGAAACACAAA GTCTACGCCTGCGAAGTCACCCATCAGGGCC TGAGCTCGCCCGTCACAAAGAGCTTCAACAG GGGAGAGTGTTGA 61 HEAVY CAGACCGTGGTGACACAGGAACCCAGCCTGA CHAIN CCGTCTCCCCTGGCGGCACCGTGACCCTGAC CD3 _((H2C)) CTGTGGAAGCAGCACAGGCGCCGTGACCAGC (VLCH1) GGCTACTACCCCAACTGGGTGCAGCAGAAGC CCGGCCAGGCCCCTAGAGGACTGATCGGCGG CACCAAGTTTCTGGCCCCTGGCACCCCCGCC AGATTCTCTGGCTCTCTGCTGGGCGGCAAGG CCGCCCTGACACTGTCTGGCGTGCAGCCTGA GGACGAGGCCGAGTACTACTGCGCCCTGTGG TACAGCAACAGATGGGTGTTCGGCGGAGGCA CCAAGCTGACCGTGCTGAGCAGC GCTAGC AC CAAGGGCCCCTCCGTGTTCCCCCTGGCCCCC AGCAGCAAGAGCACCAGCGGCGGCACAGCCG CCCTCGGCTGCCTGGTCAAGGACTACTTCCC CGAGCCCGTGACCGTGTCCTGGAACAGCGGA GCCCTGACCTCCGGCGTGCACACCTTCCCCG CCGTGCTGCAGAGCAGCGGCCTGTACAGCCT GTCCAGCGTGGTCACCGTGCCCTCCAGCAGC CTGGGCACCCAGACCTACATCTGCAACGTGA ACCACAAGCCCAGCAATACCAAGGTGGACAA GAAGGTGGAGCCCAAGAGCTGCTGA 62 FAB (MCSP)- ATGGGATGGAGCTGTATCATCCTCTTCTTGG XFAB (CD3)_((V9)) TAGCAACAGCTACCGGTGTGCATTCGGAGGT (VH-CH1-VL- GCAGCTGCAGGAAAGCGGCCCTGGCCTGGTG CH1) AAACCCAGCCAGAGCCTGAGCCTGACCTGCA GCGTGACCGGCTACAGCATCACCAGCGGCTA CTACTGGAACTGGATCAGACAGTTCCCCGGC AACAAGCTGGAATGGATGGGCTACATCACCT ACGACGGCAGCAACAACTACAACCCCAGCCT GAAGAACAGAATCAGCATCACCCGGGACACC AGCAAGAACCAGTTCTTCCTGAAGCTGAACA GCGTGACCACCGAGGACACCGCCACCTACTA CTGCGCCGACTTCGACTACTGGGGCCAGGGC ACCACCCTGACCGTGTCCTCC GCTAGC ACCA AGGGACCCAGCGTGTTCCCCCTGGCACCCAG CAGCAAGAGCACATCTGGCGGAACAGCCGCT CTGGGCTGTCTGGTGAAAGACTACTTCCCCG AGCCCGTGACCGTGTCTTGGAACTCTGGCGC CCTGACCAGCGGCGTGCACACCTTTCCAGCC GTGCTGCAGAGCAGCGGCCTGTACTCCCTGA GCAGCGTGGTGACAGTGCCCAGCAGCAGCCT GGGAACCCAGACCTACATCTGCAACGTGAAC CACAAGCCCAGCAACACCAAGGTGGACAAGA AGGTGGAACCCAAGAGCTGCGATGGCGGAGG AGGCTCCGGAGGCGGAGGCTCTGATATCCAG ATGACCCAGAGCCCCAGCTCTCTGAGCGCCA GCGTGGGCGACAGAGTGACCATCACCTGTCG GGCCAGCCAGGACATCAGAAACTACCTGAAC TGGTATCAGCAGAAGCCCGGCAAGGCCCCCA AGCTGCTGATCTACTACACCAGCAGACTGGA AAGCGGCGTGCCCTCCAGATTTTCCGGCAGC GGCTCCGGCACCGACTACACCCTGACCATCA GCAGCCTGCAGCCCGAGGATTTCGCCACATA TTACTGCCAGCAGGGCAATACCCTGCCCTGG ACCTTCGGACAGGGCACAAAAGTGGAAATCA AG 63 FAB (MCSP)- ATGGGATGGAGCTGTATCATCCTCTTCTTGG FAB (MCSP)- TAGCAACAGCTACCGGTGTGCATTCGGAGGT XFAB (CD3)_((V9)) GCAGCTGCAGGAATCTGGCCCTGGCCTGGTC (VH-CHI-VH- AAGCCAAGCCAGAGTCTGAGCCTGACCTGCA CH1-VL-CH1) GCGTGACCGGCTACAGCATTACCAGCGGCTA CTACTGGAACTGGATTCGGCAGTTCCCCGGC AATAAGCTGGAATGGATGGGCTACATCACCT ACGACGGCAGCAACAACTACAACCCCAGCCT GAAGAACCGGATCAGCATCACCCGGGACACC AGCAAGAACCAGTTCTTCCTGAAGCTGAACA GCGTGACCACCGAGGACACCGCCACATACTA TTGCGCCGACTTCGACTACTGGGGCCAGGGC ACCACCCTGACCGTGTCCAGCGCCAGCACAA AGGGCCCTAGCGTGTTCCCTCTGGCCCCCAG CAGCAAGAGCACAAGCGGCGGAACAGCCGCC CTGGGCTGCCTCGTGAAGGACTACTTCCCCG AGCCCGTGACAGTGTCTTGGAACAGCGGAGC CCTGACAAGCGGCGTGCACACCTTCCCTGCC GTGCTGCAGAGCAGCGGCCTGTACTCCCTGA GCAGCGTGGTCACCGTGCCTAGCAGCAGCCT GGGCACCCAGACCTACATCTGCAACGTGAAC CACAAGCCCAGCAACACCAAAGTGGACAAGA AGGTGGAGCCCAAGAGCTGTGATGGCGGAGG AGGGTCCGGAGGCGGTGGCTCCGAGGTGCAG CTGCAGGAATCTGGCCCTGGCCTGGTCAAGC CAAGCCAGAGTCTGAGCCTGACCTGCAGCGT GACCGGCTACAGCATTACCAGCGGCTACTAC TGGAACTGGATTCGGCAGTTCCCCGGCAATA AGCTGGAATGGATGGGCTACATCACCTACGA CGGCAGCAACAACTACAACCCCAGCCTGAAG AACCGGATCAGCATCACCCGGGACACCAGC AAGAACCAGTTCTTCCTGAAGCTGAACAGCG TGACCACCGAGGACACCGCCACATACTATTG CGCCGACTTCGACTACTGGGGCCAGGGCACC ACCCTGACCGTGTCCAGCGCCAGCACAAAGG GCCCTAGCGTGTTCCCTCTGGCCCCCAGCAG CAAGAGCACAAGCGGCGGAACAGCCGCCCTG GGCTGCCTCGTGAAGGACTACTTCCCCGAGC CCGTGACAGTGTCTTGGAACAGCGGAGCCCT GACAAGCGGCGTGCACACCTTCCCTGCCGTG CTGCAGAGCAGCGGCCTGTACTCCCTGAGCA GCGTGGTCACCGTGCCTAGCAGCAGCCTGGG CACCCAGACCTACATCTGCAACGTGAACCAC AAGCCCAGCAACACCAAAGTGGACAAGAAGG TGGAGCCCAAGAGCTGTGATGGCGGAGGAGG GTCCGGCGGCGGTGGATCCGACATCCAGATG ACCCAGAGCCCCTCTAGCCTGAGCGCCAGCG TGGGCGACAGAGTGACCATCACCTGTCGGGC CAGCCAGGACATCAGAAACTACCTGAACTGG TATCAGCAGAAGCCCGGCAAGGCCCCCAAGC TGCTGATCTACTACACCTCTAGACTGGAAAG CGGCGTGCCCAGCCGGTTTAGCGGCAGCGGC TCCGGCACCGACTACACCCTGACCATCAGCA GCCTGCAGCCCGAGGACTTCGCCACCTACTA CTGCCAGCAGGGCAACACACTCCCCTGGACC TTCGGCCAGGGCACCAAGGTGGAGATCAAGT CCAGC GCTAGC ACCAAGGGCCCCTCCGTGTT CCCCCTGGCCCCCAGCAGCAAGAGCACCAGC GGCGGCACAGCCGCCCTCGGCTGCCTGGTCA AGGACTACTTCCCCGAGCCCGTGACCGTGTC CTGGAACAGCGGAGCCCTGACCTCCGGCGTG CACACCTTCCCCGCCGTGCTGCAGAGCAGCG GCCTGTACAGCCTGTCCAGCGTGGTCACCGT GCCCTCCAGCAGCCTGGGCACCCAGACCTAC ATCTGCAACGTGAACCACAAGCCCAGCAATA CCAAGGTGGACAAGAAGGTGGAGCCCAAGAG CTGCTGA 64 FAB (MCSP)- ATGGGATGGAGCTGTATCATCCTCTTCTTGG XFAB TAGCAACAGCTACCGGTGTGCATTCGGAGGT (CD3_((V9)))- GCAGCTGCAGGAAAGCGGCCCTGGCCTGGTG FAB (MCSP) AAACCCAGCCAGAGCCTGAGCCTGACCTGCA (VH-CH1-VL- GCGTGACCGGCTACAGCATCACCAGCGGCTA CH1-VH- CTACTGGAACTGGATCAGACAGTTCCCCGGC CH1) AACAAGCTGGAATGGATGGGCTACATCACCT ACGACGGCAGCAACAACTACAACCCCAGCCT GAAGAACAGAATCAGCATCACCCGGGACACC AGCAAGAACCAGTTCTTCCTGAAGCTGAACA GCGTGACCACCGAGGACACCGCCACCTACTA CTGCGCCGACTTCGACTACTGGGGCCAGGGC ACCACCCTGACCGTGTCCTCC GCTAGC ACCA AGGGACCCAGCGTGTTCCCCCTGGCACCCAG CAGCAAGAGCACATCTGGCGGAACAGCCGCT CTGGGCTGTCTGGTGAAAGACTACTTCCCCG AGCCCGTGACCGTGTCTTGGAACTCTGGCGC CCTGACCAGCGGCGTGCACACCTTTCCAGCC GTGCTGCAGAGCAGCGGCCTGTACTCCCTGA GCAGCGTGGTGACAGTGCCCAGCAGCAGCCT GGGAACCCAGACCTACATCTGCAACGTGAAC CACAAGCCCAGCAACACCAAGGTGGACAAGA AGGTGGAACCCAAGAGCTGCGATGGCGGAGG AGGCTCCGGAGGCGGAGGCTCTGATATCCAG ATGACCCAGAGCCCCAGCTCTCTGAGCGCCA GCGTGGGCGACAGAGTGACCATCACCTGTCG GGCCAGCCAGGACATCAGAAACTACCTGAAC TGGTATCAGCAGAAGCCCGGCAAGGCCCCCA AGCTGCTGATCTACTACACCAGCAGACTGGA AAGCGGCGTGCCCTCCAGATTTTCCGGCAGC GGCTCCGGCACCGACTACACCCTGACCATCA GCAGCCTGCAGCCCGAGGATTTCGCCACATA TTACTGCCAGCAGGGCAATACCCTGCCCTGG ACCTTCGGACAGGGCACAAAAGTGGAAATCA AG AGCAGC GCTTCCACCAAAGGCCCTTCCGT GTTTCCTCTGGCTCCTAGCTCCAAGTCCACC TCTGGAGGCACCGCTGCTCTCGGATGCCTCG TGAAGGATTATTTTCCTGAGCCTGTGACAGT GTCCTGGAATAGCGGAGCACTGACCTCTGGA GTGCATACTTTCCCCGCTGTGCTGCAGTCCT CTGGACTGTACAGCCTGAGCAGCGTGGTGAC AGTGCCCAGCAGCAGCCTGGGCACCCAGACC TACATCTGCAACGTGAACCACAAGCCCAGCA ACACCAAGGTGGACAAGAAGGTGGAACCCAA GTCTTGTGGCGGAGGCGGATCCGGCGGAGGG GGATCTGAGGTGCAGCTGCAGGAAAGCGGCC CTGGCCTGGTGAAACCCAGCCAGAGCCTGAG CCTGACCTGCAGCGTGACCGGCTACAGCATC ACCAGCGGCTACTACTGGAACTGGATCAGAC AGTTCCCCGGCAACAAGCTGGAATGGATGGG CTACATCACCTACGACGGCAGCAACAACTAC AACCCCAGCCTGAAGAACAGAATCAGCATCA CCCGGGACACCAGCAAGAACCAGTTCTTCCT GAAGCTGAACAGCGTGACCACCGAGGACACC GCCACCTACTACTGCGCCGACTTCGACTACT GGGGCCAGGGCACCACCCTGACCGTGTCCTC CGCCTCTACCAAGGGCCCCAGCGTGTTCCCC CTGGCACCCAGCAGCAAGAGCACATCTGGCG GAACAGCCGCTCTGGGCTGTCTGGTGAAAGA CTACTTCCCCGAGCCCGTGACCGTGTCTTGG AACTCTGGCGCCCTGACCAGCGGCGTGCACA CCTTTCCAGCCGTGCTGCAGAGCAGCGGCCT GTACTCCCTGTCCTCCGTGGTCACCGTGCCC TCTAGCTCCCTGGGAACACAGACATATATCT GTAATGTCAATCACAAGCCTTCCAACACCAA AGTCGATAAGAAAGTCGAGCCCAAGAGCTGC TGA 65 FAB (MCSP)- ATGGGATGGAGCTGTATCATCCTCTTCTTGG FAB (MCSP)- TAGCAACAGCTACCGGTGTGCATTCGGAGGT XFAB GCAGCTGCAGGAATCTGGCCCTGGCCTGGTC (CD3_((H2C))) AAGCCAAGCCAGAGTCTGAGCCTGACCTGCA (VH-CH1-VH- GCGTGACCGGCTACAGCATTACCAGCGGCTA CH1-VL-CH1) CTACTGGAACTGGATTCGGCAGTTCCCCGGC AATAAGCTGGAATGGATGGGCTACATCACCT ACGACGGCAGCAACAACTACAACCCCAGCCT GAAGAACCGGATCAGCATCACCCGGGACACC AGCAAGAACCAGTTCTTCCTGAAGCTGAACA GCGTGACCACCGAGGACACCGCCACATACTA TTGCGCCGACTTCGACTACTGGGGCCAGGGC ACCACCCTGACCGTGTCCAGCGCCAGCACAA AGGGCCCTAGCGTGTTCCCTCTGGCCCCCAG CAGCAAGAGCACAAGCGGCGGAACAGCCGCC CTGGGCTGCCTCGTGAAGGACTACTTCCCCG AGCCCGTGACAGTGTCTTGGAACAGCGGAGC CCTGACAAGCGGCGTGCACACCTTCCCTGCC GTGCTGCAGAGCAGCGGCCTGTACTCCCTGA GCAGCGTGGTCACCGTGCCTAGCAGCAGCCT GGGCACCCAGACCTACATCTGCAACGTGAAC CACAAGCCCAGCAACACCAAAGTGGACAAGA AGGTGGAGCCCAAGAGCTGTGATGGCGGAGG AGGGTCCGGAGGCGGTGGCTCCGAGGTGCAG CTGCAGGAATCTGGCCCTGGCCTGGTCAAGC CAAGCCAGAGTCTGAGCCTGACCTGCAGCGT GACCGGCTACAGCATTACCAGCGGCTACTAC TGGAACTGGATTCGGCAGTTCCCCGGCAATA AGCTGGAATGGATGGGCTACATCACCTACGA CGGCAGCAACAACTACAACCCCAGCCTGAAG AACCGGATCAGCATCACCCGGGACACCAGCA AGAACCAGTTCTTCCTGAAGCTGAACAGCGT GACCACCGAGGACACCGCCACATACTATTGC GCCGACTTCGACTACTGGGGCCAGGGCACCA CCCTGACCGTGTCCAGCGCCAGCACAAAGGG CCCTAGCGTGTTCCCTCTGGCCCCCAGCAGC AAGAGCACAAGCGGCGGAACAGCCGCCCTGG GCTGCCTCGTGAAGGACTACTTCCCCGAGCC CGTGACAGTGTCTTGGAACAGCGGAGCCCTG ACAAGCGGCGTGCACACCTTCCCTGCCGTGC TGCAGAGCAGCGGCCTGTACTCCCTGAGCAG CGTGGTCACCGTGCCTAGCAGCAGCCTGGGC ACCCAGACCTACATCTGCAACGTGAACCACA AGCCCAGCAACACCAAAGTGGACAAGAAGGT GGAGCCCAAGAGCTGTGATGGCGGAGGAGGG TCCGGCGGCGGTGGATCCCAGACCGTGGTGA CACAGGAACCCAGCCTGACCGTCTCCCCTGG CGGCACCGTGACCCTGACCTGTGGAAGCAGC ACAGGCGCCGTGACCAGCGGCTACTACCCCA ACTGGGTGCAGCAGAAGCCCGGCCAGGCCCC TAGAGGACTGATCGGCGGCACCAAGTTTCTG GCCCCTGGCACCCCCGCCAGATTCTCTGGCT CTCTGCTGGGCGGCAAGGCCGCCCTGACACT GTCTGGCGTGCAGCCTGAGGACGAGGCCGAG TACTACTGCGCCCTGTGGTACAGCAACAGAT GGGTGTTCGGCGGAGGCACCAAGCTGACCGT GCTGAGCAGC GCTAGC ACCAAGGGCCCCTCC GTGTTCCCCCTGGCCCCCAGCAGCAAGAGCA CCAGCGGCGGCACAGCCGCCCTCGGCTGCCT GGTCAAGGACTACTTCCCCGAGCCCGTGACC GTGTCCTGGAACAGCGGAGCCCTGACCTCCG GCGTGCACACCTTCCCCGCCGTGCTGCAGAG CAGCGGCCTGTACAGCCTGTCCAGCGTGGTC ACCGTGCCCTCCAGCAGCCTGGGCACCCAGA CCTACATCTGCAACGTGAACCACAAGCCCAG CAATACCAAGGTGGACAAGAAGGTGGAGCCC AAGAGCTGCTGA 66 Murine LIGHT ATGGGATGGAGCTGTATCATCCTCTTCTTGG CHAIN TAGCAACAGCTACCGGTGTGCATTCCGAGGT CD3 _((2C11)) GCAGCTGGTGGAAAGCGGCGGAGGCCTGGTG (VHCL) CAGCCCGGCAAGAGCCTGAAGCTGAGCTGCG AGGCCAGCGGCTTCACCTTCAGCGGCTACGG CATGCACTGGGTGAGACAGGCCCCTGGCAGA GGACTGGAAAGCGTGGCCTACATCACCAGCA GCAGCATCAACATTAAGTACGCCGACGCCGT GAAGGGCCGGTTCACCGTGTCCAGGGATAAC GCCAAGAACCTGCTGTTCCTGCAGATGAACA TCCTGAAGTCCGAGGACACCGCTATGTATTA CTGCGCCAGATTCGACTGGGACAAGAACTAC TGGGGCCAGGGCACCATGGTCACAGTGTCTA GCGCTAGCGTGGCTGCACCATCTGTCTTCAT CTTCCCGCCATCTGATGAGCAGTTGAAATCT GGAACTGCCTCTGTTGTGTGCCTGCTGAATA ACTTCTATCCCAGAGAGGCCAAAGTACAGTG GAAGGTGGATAACGCCCTCCAATCGGGTAAC TCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCT GACGCTGAGCAAAGCAGACTACGAGAAACAC AAAGTCTACGCCTGCGAAGTCACCCATCAGG GCCTGAGCTCGCCCGTCACAAAGAGCTTCAA CAGGGGAGAGTGTTGA 67 Murine ATGGGATGGAGCTGTATCATCCTCTTCTTGG XFAB TAGCAACAGCTACCGGTGTGCATTCCGACAT (CD3_((2C11)))- CCAGATGACCCAGAGCCCCAGCAGCCTGCCT FAB (MCSP)- GCCAGCCTGGGCGACAGAGTGACCATCAACT FAB (MCSP) GCCAGGCCAGCCAGGACATCAGCAACTACCT (VL-CH1-VH- GAACTGGTATCAGCAGAAGCCTGGCAAGGCC CH1-VH- CCCAAGCTGCTGATCTACTACACCAACAAGC CH1) TGGCCGACGGCGTGCCCAGCAGATTCAGCGG CAGCGGCTCCGGCAGAGACAGCAGCTTCACC ATCTCCAGCCTGGAAAGCGAGGACATCGGCA GCTACTACTGCCAGCAGTACTACAACTACCC CTGGACCTTCGGCCCTGGCACCAAGCTGGAA ATCAAGAGCAGCGCTTCCACCAAAGGCCCTT CCGTGTTTCCTCTGGCTCCTAGCTCCAAGTC CACCTCTGGAGGCACCGCTGCTCTCGGATGC CTCGTGAAGGATTATTTTCCTGAGCCTGTGA CAGTGTCCTGGAATAGCGGAGCACTGACCTC TGGAGTGCATACTTTCCCCGCTGTGCTGCAG TCCTCTGGACTGTACAGCCTGAGCAGCGTGG TGACAGTGCCCAGCAGCAGCCTGGGCACCCA GACCTACATCTGCAACGTGAACCACAAGCCC AGCAACACCAAGGTGGACAAGAAGGTGGAAC CCAAGTCTTGTGGCGGAGGCGGATCCGGCGG AGGAGGGTCCGAGGTGCAGCTGCAGGAATCT GGCCCTGGCCTGGTCAAGCCAAGCCAGAGTC TGAGCCTGACCTGCAGCGTGACCGGCTACAG CATTACCAGCGGCTACTACTGGAACTGGATT CGGCAGTTCCCCGGCAATAAGCTGGAATGGA TGGGCTACATCACCTACGACGGCAGCAACAA CTACAACCCCAGCCTGAAGAACCGGATCAGC ATCACCCGGGACACCAGCAAGAACCAGTTCT TCCTGAAGCTGAACAGCGTGACCACCGAGGA CACCGCCACATACTATTGCGCCGACTTCGAC TACTGGGGCCAGGGCACCACCCTGACCGTGT CCAGCGCCAGCACAAAGGGCCCTAGCGTGTT CCCTCTGGCCCCCAGCAGCAAGAGCACAAGC GGCGGAACAGCCGCCCTGGGCTGCCTCGTGA AGGACTACTTCCCCGAGCCCGTGACAGTGTC TTGGAACAGCGGAGCCCTGACAAGCGGCGTG CACACCTTCCCTGCCGTGCTGCAGAGCAGCG GCCTGTACTCCCTGAGCAGCGTGGTCACCGT GCCTAGCAGCAGCCTGGGCACCCAGACCTAC ATCTGCAACGTGAACCACAAGCCCAGCAACA CCAAAGTGGACAAGAAGGTGGAGCCCAAGAG CTGTGATGGCGGAGGAGGGTCCGGAGGCGGT GGCTCCGAGGTGCAGCTGCAGGAATCTGGCC CTGGCCTGGTCAAGCCAAGCCAGAGTCTGAG CCTGACCTGCAGCGTGACCGGCTACAGCATT ACCAGCGGCTACTACTGGAACTGGATTCGGC AGTTCCCCGGCAATAAGCTGGAATGGATGGG CTACATCACCTACGACGGCAGCAACAACTAC AACCCCAGCCTGAAGAACCGGATCAGCATCA CCCGGGACACCAGCAAGAACCAGTTCTTCCT GAAGCTGAACAGCGTGACCACCGAGGACACC GCCACATACTATTGCGCCGACTTCGACTACT GGGGCCAGGGCACCACCCTGACCGTGTCCAG CGCCAGCACAAAGGGCCCTAGCGTGTTCCCT CTGGCCCCCAGCAGCAAGAGCACAAGCGGCG GAACAGCCGCCCTGGGCTGCCTCGTGAAGGA CTACTTCCCCGAGCCCGTGACAGTGTCTTGG AACAGCGGAGCCCTGACAAGCGGCGTGCACA CCTTCCCTGCCGTGCTGCAGAGCAGCGGCCT GTACTCCCTGAGCAGCGTGGTCACCGTGCCT AGCAGCAGCCTGGGCACCCAGACCTACATCT GCAACGTGAACCACAAGCCCAGCAACACCAA AGTGGACAAGAAGGTGGAGCCCAAGAGCTGT GATTGA 104 Light Chain ATGGGATGGAGCTGTATCATCCTCTTCTTGG antiCD33 TAGCAACAGCTACCGGTGTGCATTCCGACAT CCAGATGACCCAGAGCCCCAGCAGCCTGAGC GCCAGCGTGGGCGACAGAGTGACCATCACCT GTCGGGCCAGCGAGAGCGTGGACAACTACGG CATCAGCTTCATGAACTGGTTCCAGCAGAAG CCCGGCAAGGCCCCCAAGCTGCTGATCTACG CCGCCAGCAATCAGGGCAGCGGCGTGCCCAG CAGATTCAGCGGCTCTGGCAGCGGCACCGAC TTCACCCTGACCATCAGCAGCCTGCAGCCCG ACGACTTCGCCACCTACTACTGCCAGCAGAG CAAAGAGGTGCCCTGGACCTTCGGCCAGGGC ACCAAGGTGGAAATCAAGCGTACGGTGGCTG CACCATCTGTCTTCATCTTCCCGCCATCTGA TGAGCAGTTGAAATCTGGAACTGCCTCTGTT GTGTGCCTGCTGAATAACTTCTATCCCAGAG AGGCCAAAGTACAGTGGAAGGTGGATAACGC CCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACA GCCTCAGCAGCACCCTGACGCTGAGCAAAGC AGACTACGAGAAACACAAAGTCTACGCCTGC GAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAGAGCTTCAACAGGGGAGAGTGTTA G 105 Light Chain ATGGGATGGAGCTGTATCATCCTCTTCTTGG (CD3)_((V9)) TAGCAACAGCTACCGGTGTGCATTCCGAGGT (VH-CL) GCAGCTGGTCGAGAGCGGAGGCGGCCTGGTG CAGCCTGGCGGCAGCCTGAGACTGAGCTGCG CCGCCAGCGGCTACAGCTTCACCGGCTACAC CATGAACTGGGTCCGGCAGGCACCTGGCAAG GGACTGGAATGGGTGGCCCTGATCAACCCCT ACAAGGGCGTGAGCACCTACAACCAGAAGTT CAAGGACCGGTTCACCATCAGCGTGGACAAG AGCAAGAACACCGCCTATCTGCAGATGAACA GCCTGCGGGCCGAGGACACCGCCGTGTACTA CTGCGCCAGAAGCGGCTACTACGGCGACAGC GACTGGTACTTCGACGTGTGGGGCCAGGGCA CCCTCGTGACCGTGTCTAGCGCTAGCGTGGC TGCACCATCTGTCTTCATCTTCCCGCCATCT GATGAGCAGTTGAAATCTGGAACTGCCTCTG TTGTGTGCCTGCTGAATAACTTCTATCCCAG AGAGGCCAAAGTACAGTGGAAGGTGGATAAC GCCCTCCAATCGGGTAACTCCCAGGAGAGTG TCACAGAGCAGGACAGCAAGGACAGCACCTA CAGCCTCAGCAGCACCCTGACGCTGAGCAAA GCAGACTACGAGAAACACAAAGTCTACGCCT GCGAAGTCACCCATCAGGGCCTGAGCTCGCC CGTCACAAAGAGCTTCAACAGGGGAGAGTGT TGA 106 Fab (CD33)- ATGGGATGGAGCTGTATCATCCTCTTCTTGG CrossFab TAGCAACAGCTACCGGTGTGCATTCCCAGGT (CD3_((V9))) GCAGCTGGTGCAGTCTGGCGCCGAAGTGAAG (VH-CH1-VL- AAACCCGGCAGCAGCGTGAAGGTGTCCTGCA CH1) AGGCCAGCGGCTACACCTTCACCGACTACAA CATGCACTGGGTCCGCCAGGCCCCAGGCCAG GGACTGGAATGGATCGGCTACATCTACCCCT ACAACGGCGGCACCGGCTACAACCAGAAGTT CAAGAGCAAGGCCACCATCACCGCCGACGAG AGCACCAACACCGCCTACATGGAACTGAGCA GCCTGCGGAGCGAGGACACCGCCGTGTACTA CTGCGCCAGAGGCAGACCCGCCATGGACTAC TGGGGCCAGGGCACCCTGGTGACAGTGTCCA GCGCCAGCACAAAGGGCCCTAGCGTGTTCCC TCTGGCCCCCAGCAGCAAGAGCACAAGCGGC GGAACAGCCGCCCTGGGCTGCCTCGTGAAGG ACTACTTCCCCGAGCCCGTGACAGTGTCTTG GAACAGCGGAGCCCTGACAAGCGGCGTGCAC ACCTTCCCTGCCGTGCTGCAGAGCAGCGGCC TGTACTCCCTGAGCAGCGTGGTCACCGTGCC TAGCAGCAGCCTGGGCACCCAGACCTACATC TGCAACGTGAACCACAAGCCCAGCAACACCA AAGTGGACAAGAAGGTGGAGCCCAAGAGCTG TGATGGCGGAGGAGGGTCCGGAGGCGGTGGA TCCGACATCCAGATGACCCAGAGCCCCTCTA GCCTGAGCGCCAGCGTGGGCGACAGAGTGAC CATCACCTGTCGGGCCAGCCAGGACATCAGA AACTACCTGAACTGGTATCAGCAGAAGCCCG GCAAGGCCCCCAAGCTGCTGATCTACTACAC CTCTAGACTGGAAAGCGGCGTGCCCAGCCGG TTTAGCGGCAGCGGCTCCGGCACCGACTACA CCCTGACCATCAGCAGCCTGCAGCCCGAGGA CTTCGCCACCTACTACTGCCAGCAGGGCAAC ACACTCCCCTGGACCTTCGGCCAGGGCACCA AGGTGGAGATCAAGTCCAGCGCTAGCACCAA GGGCCCCTCCGTGTTCCCCCTGGCCCCCAGC AGCAAGAGCACCAGCGGCGGCACAGCCGCCC TCGGCTGCCTGGTCAAGGACTACTTCCCCGA GCCCGTGACCGTGTCCTGGAACAGCGGAGCC CTGACCTCCGGCGTGCACACCTTCCCCGCCG TGCTGCAGAGCAGCGGCCTGTACAGCCTGTC CAGCGTGGTCACCGTGCCCTCCAGCAGCCTG GGCACCCAGACCTACATCTGCAACGTGAACC ACAAGCCCAGCAATACCAAGGTGGACAAGAA GGTGGAGCCCAAGAGCTGCTGA 107 MCSP CDR1 GGCTACTCCATCACCAGTGGTTATTACTGGA VH AC 108 MCSP CDR2 TACATAACCTACGACGGTAGCAATAACTACA VH ACCCATCTCTCAAAAAT 109 MCSP CDR3 TTTGACTAC VH 110 MCSP CDR1 AGTGCAAGTCAGGGCATTAGAAATTATTTAA VL AC 111 MCSP CDR2 TACACATCAAGTTTACACTCA VL 112 MCSP CAGCAGTATAGTAAGCTTCCTTGGACG CDR3VL 113 GA201 CDR1 GACTACAAGATACAC VH 114 GA201 CDR2 TATTTCAACCCTAACAGCGGTTATAGTACCT VH ACGCACAGAAGTTCCAGGGC 115 GA201 CDR3 CTATCCCCAGGCGGTTACTATGTTATGGATG VH CC 116 GA201 CDR1 CGGGCAAGTCAGGGCATTAACAATTACTTAA VL AT 117 GA201 CDR2 AATACCAACAACTTGCAGACA VL 118 GA201 CDR3 TTGCAGCATAATAGTTTTCCCACG VL 119 GA201 VH CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGG TGAAGAAGCCTGGGTCCTCGGTGAAGGTCTC CTGCAAGGCCTCTGGTTTCACATTCACTGAC TACAAGATACACTGGGTGCGACAGGCCCCTG GACAAGGGCTCGAGTGGATGGGATATTTCAA CCCTAACAGCGGTTATAGTACCTACGCACAG AAGTTCCAGGGCAGGGTCACCATTACCGCGG ACAAATCCACGAGCACAGCCTACATGGAGCT GAGCAGCCTGAGATCTGAGGACACGGCCGTG TATTACTGTGCGAGACTATCCCCAGGCGGTT ACTATGTTATGGATGCCTGGGGCCAAGGGAC CACCGTGACCGTCTCCTCA 120 GA201 VL GATATCCAGATGACCCAGTCTCCATCCTCCC TGTCTGCATCTGTCGGAGACCGGGTCACCAT CACCTGCCGGGCAAGTCAGGGCATTAACAAT TACTTAAATTGGTACCAGCAGAAGCCAGGGA AAGCCCCTAAGCGCCTGATCTATAATACCAA CAACTTGCAGACAGGCGTCCCATCAAGGTTC AGCGGCAGTGGATCCGGGACAGAATTCACTC TCACCATCAGCAGCCTGCAGCCTGAAGATTT TGCCACCTATTACTGCTTGCAGCATAATAGT TTTCCCACGTTTGGCCAGGGCACCAAGCTCG AGATCAAG 121 3F2 CDR1 VH AGCTACGCCATGAGC 122 3F2 CDR2 VH GCCATCTCCGGCAGCGGAGGCAGCACCTACT ACGCCGACAGCGTGAAG 123 3F2 CDR3 VH TATTGCGCCAAGGGATGGTTCGGC 124 3F2 CDR1 VL AGAGCCAGCCAGAGCGTGACCAGCAGCTACC TG 125 3F2 CDR2 VL AACGTGGGCAGCAGACGGGCC 126 3F2 CDR3 VL TGCCAGCAGGGCATCATGCTGCCCCCC 127 3F2 VH GAGGTGCAGCTGCTGGAATCTGGAGGCGGCC TGGTGCAGCCTGGCGGCAGCCTGAGACTGTC TTGCGCCGCCAGCGGCTTCACCTTCAGCAGC TACGCCATGAGCTGGGTCCGACAGGCTCCTG GCAAGGGACTGGAATGGGTGTCCGCCATCTC CGGCAGCGGAGGCAGCACCTACTACGCCGAC AGCGTGAAGGGCCGGTTCACCATCAGCAGAG ACAACAGCAAGAACACCCTGTACCTGCAGAT GAACAGCCTGCGGGCCGAGGATACCGCCGTG TATTATTGCGCCAAGGGATGGTTCGGCGGCT TCAACTACTGGGGCCAGGGAACCCTGGTGAC AGTGTCCAGC 128 3F2 VL GAGATCGTGCTGACCCAGTCTCCCGGCACCC TGAGCCTGAGCCCTGGCGAGAGAGCCACCCT GAGCTGCAGAGCCAGCCAGAGCGTGACCAGC AGCTACCTGGCCTGGTATCAGCAGAAGCCCG GCCAGGCCCCCAGACTGCTGATCAACGTGGG CAGCAGACGGGCCACCGGCATCCCCGATAGA TTCAGCGGCAGCGGCTCCGGCACCGACTTCA CCCTGACCATCAGCCGGCTGGAACCCGAGGA CTTCGCCGTGTACTACTGCCAGCAGGGCATC ATGCTGCCCCCCACCTTCGGCCAGGGCACCA AGGTGGAAATCAAG 129 CH1A1A CDR1 GAGTTCGGCATGAAC VH 130 CH1A1A CDR2 TGGATCAACACCAAGACCGGCGAGGCCACCT VH ACGTGGAAGAGTTCAAGGGC 131 CH1A1A CDR3 TGGGACTTCGCCTATTACGTGGAAGCCATGG VH ACTAC 132 CH1A1A CDR1 AAGGCCAGTGCGGCTGTGGGTACGTATGTTG VL CG 133 CH1A1A CDR2 TCGGCATCCTACCGCAAAAGG VL 134 CH1A1A CDR3 CACCAATATTACACCTATCCTCTATTCACG VL 135 CH1A1A VH CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAG TGAAGAAACCTGGAGCTAGTGTGAAGGTGTC CTGCAAGGCCAGCGGCTACACCTTCACCGAG TTCGGCATGAACTGGGTCCGACAGGCTCCAG GCCAGGGCCTCGAATGGATGGGCTGGATCAA CACCAAGACCGGCGAGGCCACCTACGTGGAA GAGTTCAAGGGCAGAGTGACCTTCACCACGG ACACCAGCACCAGCACCGCCTACATGGAACT GCGGAGCCTGAGAAGCGACGACACCGCCGTG TACTACTGCGCCAGATGGGACTTCGCCTATT ACGTGGAAGCCATGGACTACTGGGGCCAGGG CACCACCGTGACCGTGTCTAGC 136 CH1A1A VL GATATCCAGATGACCCAGTCTCCATCCTCCC TGTCTGCATCTGTGGGAGACAGAGTCACCAT CACTTGCAAGGCCAGTGCGGCTGTGGGTACG TATGTTGCGTGGTATCAGCAGAAACCAGGGA AAGCACCTAAGCTCCTGATCTATTCGGCATC CTACCGCAAAAGGGGAGTCCCATCAAGGTTC AGTGGCAGTGGATCTGGGACAGATTTCACTC TCACCATCAGCAGTCTGCAACCTGAAGATTT CGCAACTTACTACTGTCACCAATATTACACC TATCCTCTATTCACGTTTGGCCAGGGCACCA AGCTCGAGATCAAG 137 Anti-CD33 GGCTACACCATCACCGACAGCAACATCCAC CDR1 VH 138 Anti-CD33 TACATCTACCCCTACAACGGCGGCACCGACT CDR2 VH ACAACCAG 139 Anti-CD33 GGCAACCCCTGGCTGGCCTAT CDR3 VH 140 Anti-CD33 CGGGCCAGCGAGAGCCTGGACAACTACGGCA CDR1 VL TCCGGTTTCTGACC 141 Anti-CD33 GCCGCCAGCAACCAGGGCAGC CDR2 VL 142 Anti-CD33 CAGCAGACCAAAGAGGTGCCCTGGTCC CDR3 VL 143 Anti-CD33 VH GAAGTGCAGCTGGTGCAGTCTGGCGCCGAAG TGAAGAAACCCGGCAGCAGCGTGAAGGTGTC CTGCAAGGCCAGCGGCTACACCATCACCGAC AGCAACATCCACTGGGTCCGACAGGCCCCTG GGCAGAGCCTGGAATGGATCGGCTACATCTA CCCCTACAACGGCGGCACCGACTACAACCAG AAGTTCAAGAACCGGGCCACCCTGACCGTGG ACAACCCCACCAACACCGCCTACATGGAACT GAGCAGCCTGCGGAGCGAGGACACCGCCTTC TACTACTGCGTGAACGGCAACCCCTGGCTGG CCTATTGGGGCCAGGGAACCCTGGTCACCGT GTCTAGC 144 Anti-CD33 VL GACATCCAGCTGACCCAGAGCCCCAGCACCC TGTCTGCCAGCGTGGGCGACAGAGTGACCAT CACCTGTCGGGCCAGCGAGAGCCTGGACAAC TACGGCATCCGGTTTCTGACCTGGTTCCAGC AGAAGCCCGGCAAGGCCCCCAAGCTGCTGAT GTACGCCGCCAGCAACCAGGGCAGCGGCGTG CCAAGCAGATTCAGCGGCAGCGGCTCCGGCA CCGAGTTCACCCTGACCATCAGCAGCCTGCA GCCCGACGACTTCGCCACCTACTACTGCCAG CAGACCAAAGAGGTGCCCTGGTCCTTCGGCC AGGGCACCAAGGTGGAAGTGAAG 150 (scFv)2 ATGGGCTGGTCCTGCATCATCCTGTTTCTGG antiMCSP/anti TGGCCACAGCCACCGGTGTGCATTCCGACAT huCD3 CGTGCTGACCCAGAGCCCCAGCAGCCTGAGC (LC007(VL- GCCAGCCTGGGCGACAGAGTGACCATCAGCT VH)-V9(VH- GCAGCGCCTCCCAGGGCATCAGAAACTACCT VL)) GAACTGGTATCAGCAGCGGCCCGACGGCACC GTGAAGCTGCTGATCTACTACACCAGCTCCC TGCACAGCGGCGTGCCCAGCAGATTTTCAGG CAGCGGCAGCGGCACTGACTACAGCCTGACC ATCTCCAACCTGGAACCCGAGGACATTGCCA CCTACTACTGCCAGCAGTACAGCAAGCTGCC CTGGACCTTCGGCGGAGGCACCAAGCTGGAA ATCAAGGGCGGAGGCGGATCCGGCGGAGGTG GAAGTGGCGGCGGAGGCTCTGAGGTGCAATT GCAGGAAAGCGGCCCTGGCCTGGTGAAACCC AGCCAGAGCCTGAGCCTGACCTGCAGCGTGA CCGGCTACTCCATCACCAGCGGCTACTACTG GAACTGGATCAGACAGTTCCCCGGAAACAAG CTGGAATGGATGGGCTACATCACCTACGACG GCAGCAACAACTACAACCCCAGCCTGAAGAA CCGGATCAGCATCACCCGGGACACCAGCAAG AACCAGTTCTTCCTGAAGCTGAACAGCGTGA CCACCGAGGATACCGCCACCTATTACTGTGC CGACTTCGACTACTGGGGCCAGGGCACCACC CTGACCGTGTCATCCGGTGGCGGCGGATCCG AAGTGCAGCTGGTGGAGTCTGGCGGTGGACT GGTGCAGCCAGGCGGCTCCCTGAGACTGAGC TGCGCCGCCTCCGGCTACAGCTTCACCGGCT ACACCATGAATTGGGTCCGCCAGGCCCCTGG AAAGGGACTGGAATGGGTGGCCCTGATCAAC CCCTACAAGGGCGTGAGCACCTACAACCAGA AGTTCAAGGACCGGTTCACCATCAGCGTGGA CAAGAGCAAGAACACAGCCTACCTGCAGATG AACTCCCTGAGAGCCGAGGATACCGCCGTGT ATTACTGTGCCCGCAGCGGCTACTACGGCGA CTCCGACTGGTACTTCGACGTGTGGGGGCAG GGAACCCTGGTCACCGTGTCCAGCGTGGAAG GCGGCAGCGGAGGATCTGGCGGCTCTGGCGG AAGCGGCGGAGTGGACGATATCCAGATGACA CAGTCCCCCAGCTCCCTGAGCGCCAGCGTGG GCGACAGAGTGACCATCACCTGTCGGGCCAG CCAGGACATCCGGAATTATCTCAATTGGTAT CAGCAGAAACCTGGCAAAGCTCCTAAACTGC TGATCTACTACACCTCCCGGCTGGAAAGCGG CGTGCCCAGCAGATTTTCCGGCAGCGGGAGC GGCACCGATTACACACTGACCATCAGCAGCC TGCAGCCCGAGGACTTTGCCACCTACTATTG CCAGCAGGGCAACACCCTGCCCTGGACCTTT GGGCAGGGCACAAAGGTGGAGATCAAGCACC ACCACCATCACCACTGA 154 Light Chain ATGGGATGGAGCTGTATCATCCTCTTCTTGG antiCD33 TAGCAACAGCTACCGGTGTGCATTCCGACAT _((Myelotarg)) CCAGCTGACCCAGAGCCCCTCCACACTCTCT GCCTCAGTGGGCGATAGGGTCACCATTACTT GCAGAGCTAGCGAGTCCCTGGACAACTACGG AATCCGCTTCCTTACATGGTTTCAGCAGAAG CCTGGAAAAGCACCAAAGCTGCTCATGTATG CCGCTTCTAATCAAGGCAGTGGTGTGCCCAG CCGGTTCTCCGGGTCTGGCTCAGGAACCGAA TTTACTCTGACCATTAGCTCCTTGCAGCCTG ATGACTTCGCAACATACTATTGTCAGCAGAC CAAGGAGGTCCCATGGTCTTTTGGTCAAGGC ACAAAAGTGGAGGTTAAGCGTACGGTGGCTG CACCATCTGTCTTCATCTTCCCGCCATCTGA TGAGCAGTTGAAATCTGGAACTGCCTCTGTT GTGTGCCTGCTGAATAACTTCTATCCCAGAG AGGCCAAAGTACAGTGGAAGGTGGATAACGC CCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACA GCCTCAGCAGCACCCTGACGCTGAGCAAAGC AGACTACGAGAAACACAAAGTCTACGCCTGC GAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAGAGCTTCAACAGGGGAGAGTGTTA G 155 Light Chain ATGGGATGGAGCTGTATCATCCTCTTCTTGG CD3_((V9)) TAGCAACAGCTACCGGTGTGCATTCCGATAT (VL-CH1) TCAGATGACCCAGAGCCCCAGCTCTCTGAGC GCCAGCGTGGGCGACAGAGTGACCATCACCT GTCGGGCCAGCCAGGACATCAGAAACTACCT GAACTGGTATCAGCAGAAGCCCGGCAAGGCC CCCAAGCTGCTGATCTACTACACCAGCAGAC TGGAAAGCGGCGTGCCCTCCAGATTTTCCGG CAGCGGCTCCGGCACCGACTACACCCTGACC ATCAGCAGCCTGCAGCCCGAGGATTTCGCCA CATATTACTGCCAGCAGGGCAATACCCTGCC CTGGACCTTCGGACAGGGCACAAAAGTGGAA ATCAAGAGCAGCGCTTCCACCAAAGGCCCTT CCGTGTTTCCTCTGGCTCCTAGCTCCAAGTC CACCTCTGGAGGCACCGCTGCTCTCGGATGC CTCGTGAAGGATTATTTTCCTGAGCCTGTGA CAGTGTCCTGGAATAGCGGAGCACTGACCTC TGGAGTGCATACTTTCCCCGCTGTGCTGCAG TCCTCTGGACTGTACAGCCTGAGCAGCGTGG TGACAGTGCCCAGCAGCAGCCTGGGCACCCA GACCTACATCTGCAACGTGAACCACAAGCCC AGCAACACCAAGGTGGACAAGAAGGTGGAAC CCAAGTCTTGTTGA 156 Fab ATGGGATGGAGCTGTATCATCCTCTTCTTGG (CD33_((Myelotarg)))- TAGCAACAGCTACCGGTGTGCATTCCGAGGT XFab (CD3_((V9))) GCAGCTGGTGCAGTCTGGCGCCGAAGTGAAG (VH-CH1- AAACCCGGCAGCAGCGTGAAGGTGTCCTGCA VH-CL) AGGCCAGCGGCTACACCATCACCGACAGCAA CATCCACTGGGTGCGCCAGGCCCCTGGCCAG TCTCTGGAATGGATCGGCTACATCTACCCCT ACAACGGCGGCACCGACTACAACCAGAAGTT CAAGAACCGGGCCACCCTGACCGTGGACAAC CCCACCAATACCGCCTACATGGAACTGAGCA GCCTGCGGAGCGAGGACACCGCCTTCTACTA CTGCGTGAACGGCAACCCCTGGCTGGCCTAT TGGGGCCAGGGAACACTCGTGACCGTGTCCA GCGCTAGCACCAAGGGCCCTAGCGTGTTCCC TCTGGCCCCTAGCAGCAAGAGCACCTCTGGC GGAACAGCCGCCCTGGGCTGCCTCGTGAAGG ACTACTTTCCCGAGCCCGTGACAGTGTCCTG GAACTCTGGCGCCCTGACAAGCGGCGTGCAC ACCTTTCCAGCCGTGCTGCAGTCTAGCGGCC TGTACAGCCTGAGCAGCGTCGTGACTGTGCC CAGCAGCAGCCTGGGAACCCAGACCTACATC TGCAACGTGAACCACAAGCCCAGCAACACCA AGGTGGACAAGAAGGTGGAACCCAAGAGCTG CGACGGCGGAGGCGGATCCGGGGGAGGGGGA TCTGAAGTGCAGCTGGTGGAAAGCGGCGGAG GCCTGGTGCAGCCTGGGGGATCTCTGAGACT GAGCTGTGCCGCCTCCGGCTACAGCTTCACC GGCTACACAATGAATTGGGTGCGGCAGGCTC CCGGCAAGGGCCTGGAATGGGTGGCCCTGAT CAACCCTTACAAGGGCGTGTCCACCTATAAT CAGAAGTTTAAGGACCGCTTCACCATCAGCG TGGACAAGTCCAAGAACACCGCCTACCTGCA GATGAACTCCCTGCGGGCCGAGGATACAGCC GTGTACTACTGTGCCAGAAGCGGCTACTACG GCGACAGCGACTGGTACTTCGACGTGTGGGG ACAGGGCACCCTGGTGACCGTGTCTAGTGCC TCTGTGGCCGCTCCCAGCGTGTTCATCTTCC CACCTAGCGACGAGCAGCTGAAGTCCGGCAC CGCTTCTGTCGTGTGCCTGCTGAACAACTTC TACCCCCGCGAGGCCAAGGTGCAGTGGAAAG TGGACAATGCCCTGCAGAGCGGCAACAGCCA GGAAAGCGTGACCGAGCAGGACAGCAAGGAC TCCACCTACAGCCTGTCCAGCACCCTGACAC TGAGCAAGGCCGACTACGAGAAGCACAAGGT GTACGCCTGCGAAGTGACCCACCAGGGCCTG TCTAGCCCCGTGACCAAGAGCTTCAACCGGG GCGAGTGCTGA 165 VLCL(MCSP)- CATTCCGAGGTGCAGCTGGTGGAATCTGGCG (G4S)2- GCGGACTGGTGCAGCCTGGCGGATCTCTGAG VHCL(CD3_(V9)) ACTGAGCTGTGCCGCCAGCGGCTACAGCTTC ACCGGCTACACCATGAACTGGGTGCGCCAGG CCCCTGGCAAGGGACTGGAATGGGTGGCCCT GATCAACCCCTACAAGGGCGTGTCCACCTAC AACCAGAAGTTCAAGGACCGGTTCACCATCA GCGTGGACAAGAGCAAGAACACCGCCTACCT GCAGATGAACAGCCTGCGGGCCGAGGACACC GCCGTGTACTATTGTGCCAGAAGCGGCTACT ACGGCGACAGCGACTGGTACTTCGACGTGTG GGGCCAGGGCACACTCGTGACCGTGTCAAGC GCTAGCGTGGCCGCTCCCAGCGTGTTCATCT TCCCACCTAGCGACGAGCAGCTGAAGTCCGG CACAGCCTCTGTCGTGTGCCTGCTGAACAAC TTCTACCCCCGCGAGGCCAAGGTGCAGTGGA AGGTGGACAATGCCCTGCAGAGCGGCAACAG CCAGGAAAGCGTGACCGAGCAGGACAGCAAG GATAGCACCTACAGCCTGAGCAGCACCCTGA CCCTGAGCAAGGCCGACTACGAGAAGCACAA GGTGTACGCCTGCGAAGTGACCCACCAGGGC CTGTCTAGCCCCGTGACCAAGAGCTTCAACC GGGGCGAGTGTGATGGCGGAGGCGGATCCGG GGGAGGCGGCTCTGATATTGTGCTGACCCAG AGCCCCAGCAGCCTGTCTGCCTCTCTGGGCG ACAGAGTGACCATCAGCTGTAGCGCCTCTCA GGGCATCCGGAACTACCTGAACTGGTATCAG CAGCGGCCCGACGGCACCGTGAAGCTGCTGA TCTACTACACCAGCTCCCTGCACTCCGGCGT GCCCAGCAGATTTTCTGGCAGCGGCTCCGGC ACCGACTACTCCCTGACCATCTCCAACCTGG AACCCGAGGATATCGCCACCTACTACTGCCA GCAGTACTCCAAGCTGCCCTGGACCTTTGGA GGCGGCACCAAGCTGGAAATCAAGCGTACGG TGGCTGCCCCCTCCGTGTTTATCTTTCCCCC ATCCGATGAACAGCTGAAAAGCGGCACCGCC AGCGTCGTGTGTCTGCTGAACAATTTTTACC CTAGGGAAGCTAAAGTGCAGTGGAAAGTGGA TAACGCACTGCAGTCCGGCAACTCCCAGGAA TCTGTGACAGAACAGGACTCTAAGGACAGCA CATACTCCCTGTCCTCCACCCTGACACTGTC TAAGGCTGATTATGAGAAACACAAAGTGTAT GCTTGTGAAGTGACACATCAGGGACTGAGCA GCCCTGTGACAAAGTCCTTCAACAGAGGCGA GTGCTGATGAA 166 VHCH1(MCSP)- ATGGGATGGAGCTGTATCATCCTCTTCTTGG (G4S)2- TAGCAACAGCTACCGGTGTGCATTCGGAGGT VLCH1(CD3_(V9)) GCAGCTGCAGGAATCTGGCCCTGGCCTGGTC AAGCCAAGCCAGAGTCTGAGCCTGACCTGCA GCGTGACCGGCTACAGCATTACCAGCGGCTA CTACTGGAACTGGATTCGGCAGTTCCCCGGC AATAAGCTGGAATGGATGGGCTACATCACCT ACGACGGCAGCAACAACTACAACCCCAGCCT GAAGAACCGGATCAGCATCACCCGGGACACC AGCAAGAACCAGTTCTTCCTGAAGCTGAACA GCGTGACCACCGAGGACACCGCCACATACTA TTGCGCCGACTTCGACTACTGGGGCCAGGGC ACCACCCTGACCGTGTCCAGCGCCAGCACAA AGGGCCCTAGCGTGTTCCCTCTGGCCCCCAG CAGCAAGAGCACAAGCGGCGGAACAGCCGCC CTGGGCTGCCTCGTGAAGGACTACTTCCCCG AGCCCGTGACAGTGTCTTGGAACAGCGGAGC CCTGACAAGCGGCGTGCACACCTTCCCTGCC GTGCTGCAGAGCAGCGGCCTGTACTCCCTGA GCAGCGTGGTCACCGTGCCTAGCAGCAGCCT GGGCACCCAGACCTACATCTGCAACGTGAAC CACAAGCCCAGCAACACCAAAGTGGACAAGA AGGTGGAGCCCAAGAGCTGTGATGGCGGAGG AGGGTCCGGAGGCGGTGGATCCGACATCCAG ATGACCCAGAGCCCCTCTAGCCTGAGCGCCA GCGTGGGCGACAGAGTGACCATCACCTGTCG GGCCAGCCAGGACATCAGAAACTACCTGAAC TGGTATCAGCAGAAGCCCGGCAAGGCCCCCA AGCTGCTGATCTACTACACCTCTAGACTGGA AAGCGGCGTGCCCAGCCGGTTTAGCGGCAGC GGCTCCGGCACCGACTACACCCTGACCATCA GCAGCCTGCAGCCCGAGGACTTCGCCACCTA CTACTGCCAGCAGGGCAACACACTCCCCTGG ACCTTCGGCCAGGGCACCAAGGTGGAGATCA AGTCCAGCGCTAGCACCAAGGGCCCCTCCGT GTTCCCCCTGGCCCCCAGCAGCAAGAGCACC AGCGGCGGCACAGCCGCCCTCGGCTGCCTGG TCAAGGACTACTTCCCCGAGCCCGTGACCGT GTCCTGGAACAGCGGAGCCCTGACCTCCGGC GTGCACACCTTCCCCGCCGTGCTGCAGAGCA GCGGCCTGTACAGCCTGTCCAGCGTGGTCAC CGTGCCCTCCAGCAGCCTGGGCACCCAGACC TACATCTGCAACGTGAACCACAAGCCCAGCA ATACCAAGGTGGACAAGAAGGTGGAGCCCAA GAGCTGCTGA

While there are shown and described presently preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims. 

The invention claimed is:
 1. A bispecific antibody comprising a first and a second Fab fragment, wherein: (a) the first Fab fragment comprises one antigen binding site specific for a tumor antigen; (b) the second Fab fragment comprises one antigen binding site specific for a CD3 T-cell co-receptor (CD3) antigen, wherein the second Fab fragment is a crossover Fab fragment; and (c) the two Fab fragments are connected to each other by two peptide linkers, wherein: (i) each peptide linker connects a C-terminus of the first Fab fragment with an N-terminus of the second Fab fragment, or (ii) each peptide linker connects an N-terminus of the first Fab fragment with a C-terminus of the second Fab fragment; and wherein the bispecific antibody is devoid of a Fc domain.
 2. The bispecific antibody of claim 1, additionally comprising a third Fab fragment.
 3. The bispecific antibody of claim 2, wherein the third Fab fragment comprises one antigen binding site specific for the tumor antigen.
 4. The bispecific antibody of claim 2 or 3, wherein the third Fab fragment is connected to the first Fab fragment by one or two peptide linkers.
 5. The bispecific antibody of claim 2 or 3, wherein the third Fab fragment is connected to the second Fab fragment by one or two peptide linkers.
 6. The bispecific antibody of any one of claim 1, 2, and 3, wherein the tumor antigen is selected from the group of melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA), fibroblast activation protein (FAP), and CD33.
 7. The bispecific antibody of claim 6, wherein the tumor antigen is MCSP.
 8. A pharmaceutical composition comprising a bispecific antibody of any one of claim 1, 2, and
 3. 9. A prokaryotic or eukaryotic host cell comprising vectors comprising nucleic acid molecules encoding the bispecific antibody of any one of claim 1, 2, and
 3. 10. A method of producing the antibody of any one of claim 1, 2, and 3 comprising culturing the host cell of claim 9 so that the antibody is produced.
 11. A bispecific antibody comprising a first and a second Fab fragment, wherein: (a) the first Fab fragment comprises one antigen binding site specific for MCSP, and the second Fab fragment comprises one antigen binding site specific for a CD3 antigen, wherein the variable heavy chain region and variable light chain region of the second Fab fragment are exchanged; (b) the two Fab fragments are connected to each other by a first and second peptide linker, wherein the first peptide linker connects the C-terminus of a constant light chain region of the first Fab fragment with the N-terminus of the variable heavy chain region of the second Fab fragment, and the second peptide linker connects the C-terminus of a constant heavy chain 1 region of the first Fab fragment with the N-terminus of the variable light chain region of the second Fab fragment; and (c) the bispecific antibody is devoid of a Fc domain.
 12. The bispecific antibody of claim 1 or 11, comprising a first polypeptide having the amino acid sequence of SEQ ID NO: 163 and a second polypeptide having the amino acid sequence of SEQ ID NO:
 164. 